Modified matrix proteins of vesicular stomatitis virus

ABSTRACT

The present invention relates to vesicular stomatitis virus (VSV) matrix (M) protein mutants. One mutant M protein includes a glycine changed to a glutamic acid at position (21), a leucine changed to alanine at position (111) and a methionine changed to an arginine at position (51). Another M protein mutant includes a glycine changed to a glutamic acid at position (22) and a methionine changed to an arginine at positions (48) and (51). These new rVSVs having the mutant M are significantly attenuated and lost virulence, including neurovirulence, and are capable of inducing an immune responses against an antigen of interest. In addition, a rVSV serotype Indiana having the first described M mutant is capable of efficient replication at 31° C., and of poor replication or incapable of replication at about 37° C. or higher.

FIELD OF THE INVENTION

The present invention relates, in general, to biotechnology andimmunology. In particular, the present invention relates to modifiedmatrix (M) proteins of vesicular stomatitis virus (VSV), to attenuatedrecombinant VSVs expressing the modified M proteins and to VSV basedprime-boost vaccines that induce long-lasting humoral, cell-mediated andmucosal immune responses against foreign antigens expressed by therecombinant VSV carrying foreign genes.

BACKGROUND OF THE INVENTION

Vesicular stomatitis virus (VSV) is a negative stranded RNA virus whichinfects most mammalian cells and expresses viral proteins up to 60% oftotal proteins in infected cells [Kim, G. N., and C. Y. Kang. Virology357:41, 2007]. In nature VSV infects pigs, cattle, and horses, andcauses vesicular disease around the mouth and foot. Although humaninfection by VSV has been reported, VSV does not cause any serioussymptoms in humans [Fields, B. N., and K. Hawkins. N Engl J Med 277:989,1967; Johnson, K. M. et al. Am J Trop Med Hyg 15:244, 1966].

VSV encodes five proteins, nucleocapsid protein (N), phosphoprotein (P),matrix protein (M), surface glycoprotein (G), and RNA dependent RNApolymerase (L). The N, P, and L proteins of VSV are required forsynthesis of positive sense and negative sense genomic RNAs and mRNA,which are necessary for the synthesis of VSV proteins.

Blocking the host cellular protein synthesis by VSV matrix (M) proteininduces cell death. Changing a methionine residue at position 51 of theM protein to arginine (M51R) in the vesicular stomatitis virus Indianaserotype (VSV_(Ind)), and changing methionines at position 48 (Met48)and 51 (Met51) to Arg in the vesicular stomatitis virus New Jerseyserotype (VSV_(NJ)) M gene could negate this function of VSV M protein(Kim, G. and Kang, C., Virology, 357:41-53, 2007). While the VSVs withthese Met to Arg mutations in the M protein have significantly reducedcytopathic effects, they still replicate and produce progeny viruses inthe cell and in the infected animal. One of the temperature sensitive(ts) M gene mutants of VSV_(Ind) Orsay strain, tsO23 has shown limitedreplication in a mouse glial cell line at 37° C. and 39° C. (Rabinowitz,S. et al. Infection and Immunity 33:120-125, 1981). It has beendemonstrated that the temperature sensitivity of tsO23 was the result ofthe improper or lack of the initiation of the viral assembly with thecharacteristic bullet shaped structure at 39° C. (Flood, E. et al.Virology 278:520-533, 2000; Lyles, D. et al. Virology, 217:76-87, 1996).In addition, the tsO23 lost its neurovirulence in mice even after directinoculation into the brain (Rabinowitz, S. et al. Infection andImmunity, 33:120-125, 1981).

There are three amino acid differences between wild type M of VSV Orsaystrain and M of tsO23, which are glycin (G) to glutamic acid (E) at 21stamino acid (G21E), leucine (L) to phenylalanine (F) at 111th amino acid(L111F), and histidine (H) to tyrosine (Y) at 227th amino acids (H227Y)(Morita, K. et al. J. Virol. 61:256-263, 1987). The single amino acidreversion at F111 to L in revertants implicated that the F111 appears toplay a major role in the temperature sensitivity of the tsO23. However,the other two mutations may also have some role because the singlereversion from the F to L at 111th position did not recover the viruscompletely to its wild type phenotype (Morita, K. et al. J. Virol.61:256-263, 1987; Li, Y. et al. J. Virol. 62:3729-3737, 1988).

Given the single amino acid reversions at F111 to L in revertants, itmay be advantageous to create mutations that are both less susceptibleto reversion and that play a role in the temperature sensitivity of thetsO23.

Currently, research groups have developed replication competent,assembly defective VSV having a G glycoprotein (G) gene deleted (ΔG) orboth G and M genes deleted (ΔMG) as safer vaccine vectors (Kahn, J. etal. J. Virol. 75:11079-11087, 2001; Schwartz, J. et al., Virology366:166-73, 2007). However, having the G gene deleted or M and G genedeleted, the VSV vector requires the supply of G or both M and Gproteins in trans for the production of the assembly-defective VSV. Inorder to reduce the cost of producing vaccines, it is necessary togenerate a system, which can produce the viral vaccine vectors that canreplicate in high titre. Therefore, what is needed is a VSV vectorsystem, which may be a full-length VSV vector, which has lost itsvirulence (avirulent) and still can replicate to a high titer in vitroat 31° C. cannot assemble properly at non-permissible temperatures,induce good immune responses against the gene of interest that itexpresses, and having less chance of reverting back to a wild typephenotype.

Further and other objects of the invention will be realized from thefollowing Summary of the Invention, the Discussion of the Invention andthe embodiments and Examples thereof.

SUMMARY OF THE INVENTION

The inventors have generated new matrix (M) protein mutants of vesicularstomatitis virus (VSV), including VSV Indiana serotype (VSV_(Ind)) andVSV New Jersey serotype (VSV_(NJ)). These new VSVs having the mutant Mproteins are essentially non-cytolytic, significantly attenuated andavirulent, including neurovirulence (safer). Furthermore, these new VSVshaving the mutant M proteins are capable of inducing immune responsesagainst an antigen or epitope of interest. The immune response may behumoral, cellular and mucosal immune responses. In addition, theVSV_(Ind) having the M mutant can assemble and be released from theinfected cells normally at 31° C., but they can not assemble properly at37° C. or higher and the release of the viruses from infected cells isreduced significantly relative to wild type VSV_(Ind). In addition, theM protein mutants of the present invention may be less susceptible toreversion relative to known M protein mutants of the prior art.

As such, in one embodiment, the present application relates to amodified matrix (M) protein of a vesicular stomatitis virus (VSV). Inone embodiment, the modified M protein of the present invention includesan amino acid sequence selected from the group consisting of: (i) SEQ IDNO: 3 including at least the following substitutions: G21E/L111A/M51R;and (ii) SEQ ID NO: 8 including at least the following substitutions:G22E/M48R/M51R. In one aspect of the present invention, SEQ ID NO:8includes at least the following substitutions: G22E/L110A/M48R/M51R.

In one embodiment of the present invention, the modified M protein of(i) includes an amino acid sequence SEQ ID NO:4 and the modified Mprotein of (ii) includes the amino acid sequence SEQ ID NO:9 or SEQ IDNO:10.

In one embodiment of the present invention, the E at position 21 in (i)and the E at position 22 in (ii) are encoded by a gaa codon, and whereinthe R at position 51 in (i) and (ii) and the R at position 48 in (ii) isencoded by a cga codon.

In one embodiment of the present invention, the A at position 111 of SEQID NO:4 and the A at position 110 of SEQ ID NO:10 is encoded by a gcacodon.

In one embodiment of the present invention, the amino acid sequence of(i) is encoded by a gene comprising SEQ ID NO:2, and the amino acidsequence of (ii) is encoded by a gene comprising SEQ ID NO:6 or SEQ IDNO:7.

In another embodiment, the present invention provides for a nucleotidesequence or a gene that encodes a modified matrix (M) protein of avesicular stomatitis virus (VSV), wherein the nucleotide sequence orgene includes a nucleotide sequence selected from SEQ ID NO:2, SEQ IDNO:6 and SEQ ID NO: 7.

In another embodiment the present invention provides for a recombinantVSV (rVSV). The rVSV of the present invention, in one embodiment,includes a nucleotide sequence or gene comprising a nucleotide sequenceselected from SEQ ID NOs:2, 6 and 7.

In another embodiment the present invention provides for a recombinantvesicular stomatitis virus (rVSV). The rVSV of the present inventionincludes a modified matrix (M) protein according to any of the previousembodiments.

In one embodiment of the present invention, the rVSV is a recombinantvesicular stomatitis virus Indiana serotype (rVSVInd), and the modifiedM protein includes the amino acid sequence of SEQ ID NO: 3 including atleast the following substitution: G21E/L111A/M51R.

In another embodiment of the present invention, the modified M proteinof the rVSV_(Ind) includes the amino acid sequence of SEQ ID NO: 4.

In another embodiment of the present invention, the modified M proteinof the rVSV_(Ind) is encoded by a gene comprising a nucleotide sequenceof SEQ ID NO: 2.

In another embodiment of the present invention, the rVSVInd is capableof producing VSV_(Ind) particles at permissible temperatures andincapable of producing the particles at non-permissible temperatures.

In another embodiment of the present invention, the rVSV is arecombinant vesicular stomatitis virus New Jersey serotype (rVSV_(NJ)),and the modified M protein includes the amino acid sequence of SEQ IDNO: 8 including at least the following substitutions: G22E/M48R/M51R. Inone aspect of this embodiment, SEQ ID NO:8 includes at least thefollowing substitutions: G22E/L110A/M48R/M51R.

In one embodiment, the modified M protein of the rVSV_(NJ) is encoded bya gene including a nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO:7.

In one embodiment, the modified M protein of the rVSV_(NJ) includes theamino acid sequence SEQ ID NO: 9 or SEQ ID NO:10.

In another embodiment of the present invention, the rVSV is a chimericrVSV that expresses a protein of a foreign pathogen.

In one embodiment of the present invention, the pathogen is a viral,fungal, bacterial or parasitic pathogen.

In another embodiment, the rVSV of the present invention is essentiallynon-cytolytic and avirulent.

In one embodiment, the present invention provides for a vaccine. Thevaccine, in one embodiment, includes an effective amount of a modifiedmatrix (M) protein, the modified M protein being encoded by a nucleotidesequence comprising a sequence selected from: SEQ ID NO:2, SEQ ID NO:6and SEQ ID NO:7.

In another embodiment, the present invention provides for a vaccine. Thevaccine, in one embodiment, includes an effective amount of one or moreattenuated recombinant vesicular stomatitis virus (rVSV), the one ormore attenuated rVSVs including a modified matrix (M) protein, themodified M protein comprising an amino acid sequence selected from thegroup consisting of: (i) SEQ ID NO: 3 including at least the followingsubstitutions: G21E/L111A/M51R, and (ii) SEQ ID NO: 8 including at leastthe following substitutions: G22E/M48R/M51R. In one aspect, SEQ ID NO:8further includes the substitution L110A.

In one embodiment of the vaccine of the present invention, the rVSV is arecombinant vesicular stomatitis virus Indiana serotype (rVSV_(Ind)),and the modified M protein includes the amino acid sequence of SEQ IDNO: 3 including at least the following substitution: G21E/L111A/M51R.

In another embodiment of the vaccine of the present invention, the rVSVis a recombinant vesicular stomatitis virus Indiana serotype(rVSV_(Ind)), and the modified M protein includes the amino acidsequence of SEQ ID NO: 4.

In another embodiment of the vaccine of the present invention, themodified M protein is encoded by a gene comprising a nucleotide sequenceof SEQ ID NO: 2.

In another embodiment of the vaccine of the present invention, therVSV_(Ind) is capable of producing rVSV_(Ind) particles at permissibletemperatures and incapable of producing the particles at non-permissibletemperatures.

In another embodiment of the vaccine of the present invention, therVSV_(Ind) is a full length rVSV_(Ind).

In another embodiment of the vaccine of the present invention, the rVSVis a recombinant vesicular stomatitis virus New Jersey serotype(rVSV_(NJ)), and the modified M protein comprises the amino acidsequence of SEQ ID NO: 8 including at least the following substitutions:G22E/M48R/M51R. In one aspect of this embodiment, SEQ ID NO:8 furtherincludes the substitution L110A.

In one embodiment of the vaccine of the present invention, the rVSV is arecombinant vesicular stomatitis virus New Jersey serotype (rVSV_(NJ)),and wherein the modified M protein comprises the amino acid sequence SEQID NO: 9 or SEQ ID NO:10.

In one embodiment of the vaccine of the present invention, the modifiedM protein is encoded by a gene having a nucleotide sequence of SEQ IDNO: 6 or SEQ ID NO:7.

In another embodiment of the vaccine of the present invention, therVSV_(NJ) is a full length rVSV_(NJ).

In another embodiment of the vaccine of the present invention, the rVSVis a chimeric rVSV that expresses a protein of a foreign pathogen, andwherein said chimeric rVSV is capable of inducing an immune response tosaid protein.

In another embodiment of the vaccine of the present invention, thevaccine comprises a mixture of attenuated chimeric rVSVs, wherein atleast two chimeric rVSVs in the mixture express a different protein ofthe foreign pathogen.

In another embodiment of the vaccine of the present invention, thepathogen is a viral, a fungal, a bacterial or a parasitic pathogen.

In another embodiment of the vaccine of the present invention, thepathogen is a lentivirus.

In another embodiment of the vaccine of the present invention, thelentivirus is a HIV and the protein of the foreign pathogen is a HIVprotein.

In another embodiment of the vaccine of the present invention, thepathogen is HCV and the protein of the foreign pathogen is a HCVprotein.

In another embodiment of the vaccine of the present invention, the E atposition 21 in (i) and the E at position 22 are encoded by a gaa codon,and the R at position 51 and the R at position 48 is encoded by a cgacodon.

In another embodiment of the vaccine of the present invention, the A atposition 111 and the A at position 110 is encoded by a gca codon.

In another embodiment of the vaccine of the present invention, vaccineis capable of inducing a humoral, cellular and mucosal immune response.

In another embodiment of the vaccine of the present invention, thevaccine further includes an adjuvant.

In one embodiment, the present invention provides for a prime boostcombination vaccine. The prime boost combination vaccine, according toone embodiment, includes: (a) an effective amount of a vaccinecomprising an attenuated recombinant vesicular stomatitis virus (rVSV)of one serotype having a first modified M protein comprising the aminoacid sequence of SEQ ID NO:4; and (b) an effective amount of a vaccinecomprising a rVSV of another serotype having a second modified M proteincomprising the amino acid sequence of SEQ ID NO:9 or comprising theamino acid sequence of SEQ ID NO:10.

In one embodiment of the prime boost combination vaccine, SEQ ID NO:4 isencoded by a gene comprising SEQ ID NO:2.

In another embodiment of the prime boost combination vaccine, SEQ IDNO:9 is encoded by a gene comprising SEQ ID NO:6 and SEQ ID NO:10 isencoded by a gene comprising SEQ ID NO:7.

In another embodiment of the prime boost combination vaccine, (a) is apriming vaccine and (b) is a booster vaccine.

In another embodiment of the prime boost combination vaccine, (b) is apriming vaccine and (a) is a booster vaccine.

In another embodiment of the prime boost combination vaccine of thepresent invention, the two attenuated rVSVs are chimeric rVSVs thatexpress a protein of a foreign pathogen, and wherein the two chimericrVSVs are capable of inducing an immune response to the protein.

In another embodiment of the prime boost combination vaccine of thepresent invention, the pathogen is a viral, a fungal, a bacterial or aparasitic pathogen.

In another embodiment of the prime boost combination vaccine of thepresent invention, the pathogen is a lentivirus.

In another embodiment of the prime boost combination vaccine of thepresent invention, the lentivirus is a HIV and the protein is a HIVprotein.

In another embodiment of the prime boost combination vaccine of thepresent invention, the rVSV of one serotype and the rVSV of the otherserotype include a surface glycoprotein (G) gene and a RNA dependent RNApolymerase (L) gene, and wherein a gene for expressing the HIV proteinis inserted in between the G gene and the L gene.

In another embodiment of the prime boost combination vaccine of thepresent invention, wherein the HIV gene is selected from the group ofHIV genes consisting of env, gag and pol.

In another embodiment of the prime boost combination vaccine of thepresent invention, the pathogen is HCV and the epitope is a HCV protein.

In another embodiment of the prime boost combination vaccine of thepresent invention, the rVSV of one serotype and the rVSV of the otherserotype include a surface glycoprotein (G) gene and a RNA dependent RNApolymerase (L) gene, and a gene for expressing the HCV protein isinserted in between the G gene and the L gene.

In another embodiment of the prime boost combination vaccine of thepresent invention, the HCV protein is a structural or a non-structuralHCV protein.

In another embodiment of the prime boost combination vaccine of thepresent invention, each one of the two vaccines includes a mixture ofthe attenuated chimeric rVSVs, and at least two of the attenuatedchimeric rVSVs in the mixture have a different protein of the pathogen.

In another embodiment of the prime boost combination vaccine of thepresent invention, each one of the two vaccines is capable of inducinghumoral, cellular and mucosal immune responses.

In another embodiment of the prime boost combination vaccine of thepresent invention, the serotype of the rVSV of vaccine (a) is Indianaand the serotype of the rVSV of vaccine (b) is New Jersey.

In another embodiment of the prime boost combination vaccine of thepresent invention, each one of vaccine (a) and vaccine (b) furthercomprises an adjuvant.

According to another embodiment, the present invention provides for akit. The kit, in one embodiment, includes: (a) at least one dose of aneffective amount of a vaccine comprising a recombinant vesicularstomatitis virus Indiana serotype (rVSV_(Ind)) having a modified Mprotein comprising the amino acid sequence of SEQ ID NO:4, and (b) atleast one dose of an effective amount of a vaccine comprising arecombinant vesicular stomatitis virus New Jersey serotype (rVSV_(NJ))having a modified M protein comprising the amino acid sequence of SEQ IDNO:9 or the amino acid sequence of SEQ ID NO:10.

In one embodiment of the kit of the present invention (a) and (b) areformulated in a pharmaceutically acceptable carrier.

In another embodiment of the kit of the present invention SEQ ID NO:4 isencoded by a gene comprising SEQ ID NO:2.

In another embodiment of the kit of the present invention SEQ ID NO:9 isencoded by a gene comprising SEQ ID NO:6 and SEQ ID NO:10 is encoded bya gene comprising SEQ ID NO:7.

In another embodiment, the present invention provides for an isolatedpeptide comprising an amino acid sequence selected from the group ofamino acid sequences listed as SEQ ID NOs: 4, 9 and 10. In oneembodiment the isolated peptide is provided in purified form.

In another embodiment, the present invention provides for an isolatednucleotide sequences comprising a nucleotide sequence selected from thegroup SEQ ID NO:2, SEQ ID NO:6 and SEQ ID NO:7. In one embodiment theisolated nucleotide sequences is provided in purified form.

In one embodiment, the present invention provides for a vaccine of thepresent invention for use to induce an immune response in a subject.

In one embodiment, the present invention relates to the prime boostcombination vaccine of the present invention for use to induce an immuneresponse in a subject.

In another embodiment, the present invention relates to a method ofinducing an immune response in a subject. The method, according to oneembodiment, includes administering to the subject: (a) an effectiveamount of a vaccine comprising an attenuated recombinant vesicularstomatitis virus (rVSV) of one serotype having a first modified Mprotein, the first modified M protein comprising the amino acid sequenceof SEQ ID NO: 3 including at least the following substitutions:G21E/L111A/M51R; and (b) an effective amount of another vaccinecomprising an attenuated rVSV of another serotype having a secondmodified M protein, the second modified M protein comprising the aminoacid sequence of SEQ ID NO: 8 including at least the followingsubstitutions: G22E/M48R/M51R. In one aspect of this embodiment, SEQ IDNO:8 further includes the substitution L110A.

In one embodiment of the method of inducing an immune response in asubject of the present invention (a) is administered to the subjectbefore (b) is administered to the subject.

In another embodiment of the method of inducing an immune response in asubject of the present invention (b) is administered to the subject morethan one time over the course of inducing.

In another embodiment of the method of inducing an immune response in asubject of the present invention (a) is administered to the subject and(b) is administered to the subject at about weeks three, eight andsixteen post-administration of (a).

In another embodiment of the method of inducing an immune response in asubject of the present invention (b) is administered to the subjectbefore (a) is administered to the subject.

In another embodiment of the method of inducing an immune response in asubject of the present invention (a) is administered to the subject morethan one time over the course of inducing.

In another embodiment of the method of inducing an immune response in asubject of the present invention (b) is administered to the subject and(a) is administered to the subject at about weeks three, eight andsixteen post-administration of (b).

In another embodiment of the method of inducing an immune response in asubject of the present invention the two rVSVs are chimeric rVSVs thatexpress a protein of a foreign pathogen, and wherein the two rVSVs arecapable of inducing an immune response to the protein.

In another embodiment of the method of inducing an immune response in asubject of the present invention the pathogen is a viral, a fungal, abacterial or a parasitic pathogen.

In another embodiment of the method of inducing an immune response in asubject of the present invention the pathogen is a lentivirus.

In another embodiment of the method of inducing an immune response in asubject of the present invention the lentivirus is a humanimmunodeficiency virus (HIV) and the protein is a HIV protein.

In another embodiment of the method of inducing an immune response in asubject of the present invention the rVSV of one serotype and the rVSVof the other serotype include a surface glycoprotein (G) gene and a RNAdependent RNA polymerase (L) gene, and a gene for expressing the HIVprotein is a HIV gene inserted in between the G gene and the L gene.

In another embodiment of the method of inducing an immune response in asubject of the present invention the HIV gene is selected from the groupof HIV genes consisting of gag, env and pol.

In another embodiment of the method of inducing an immune response in asubject of the present invention the pathogen is hepatitis C virus (HCV)and the protein is a HCV protein.

In another embodiment of the method of inducing an immune response in asubject of the present invention the rVSV of one serotype and the rVSVof the other serotype include a surface glycoprotein (G) gene and a RNAdependent RNA polymerase (L) gene, and wherein a gene for expressing theHCV protein is inserted in between the G gene and the L gene of therVSV.

In another embodiment of the method of inducing an immune response in asubject of the present invention the two vaccines comprise a mixture ofattenuated chimeric rVSVs, and at least two of the attenuated chimericrVSVs in the mixture express a different protein of the pathogen.

In another embodiment of the method of inducing an immune response in asubject of the present invention each one of the two vaccines (a) and(b) induces humoral, cellular and mucosal immune responses.

In another embodiment of the method of inducing an immune response in asubject of the present invention the one serotype of vaccine (a) isIndiana and the other serotype of vaccine (b) is New Jersey.

In another embodiment of the method of inducing an immune response in asubject of the present invention each of vaccine (a) and vaccine (b)further includes an adjuvant.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments will now be described, by way of example only, withreference to the drawings, in which:

FIG. 1 illustrates gene organization of the vesicular stomatitis virus(VSV), Indiana (Ind) serotype with mutations in the M gene.

FIG. 2 illustrates gene organization of vesicular stomatitis virus(VSV), New Jersey (NJ) with mutations in the M gene.

FIG. 3 illustrates a reverse genetics system for the recovery of VSVfrom cDNA.

FIG. 4 illustrates the burst size effect of mutation L111F in the Mprotein of rVSV_(Ind) at permissive temperature [panels A) and C)] andat semi-permissive temperature [panels B) and D)], in baby hamsterkidney (BHK) cells infected with recombinant VSVs [panels A) and B)] andin human neuroblastoma cells (SH-5Y5Y) infected with the rVSV.

FIG. 5 is a graph representing the burst size effect of mutationsM48R+M51R, G22E, G22E/M48R+M51R, G22E/L110F, G22E/L110F/M48R+M51R in theM protein of rVSV_(NJ) at 31° C. and 37° C. in BHK21 cells.

FIG. 6 A) is a Western blot analysis illustrating the level of rVSVprotein expression of rVSV_(Ind) 1. WT, 2. M51R, 3. G21E 4. G21E/M51R 5.G21E/L111F and 6. G21E/L111F/M51R.

FIG. 6 B) is a Western blot analysis illustrating the level of rVSVprotein expression of rVSV_(NJ) 1. WT, 2. M48R+M51R, 3. G22E 4.G22E/M48R+M51R 5. G22E/L110F and 6. G22E/L110F/M48R+M51R.

FIG. 7 includes photographs of BHK21 cells infected with rVSV_(Ind)having a wild type M gene (panels B and I) and with rVSV_(Ind) havingdifferent mutations in the M gene (panels A, D, E, F, G, H, K, L, M andN) and photographs of uninfected BHK21 cells (panels C and J). Cellswere incubated at 31° C. (panels A to G) or 37° C. (panels H to N).

FIG. 8 includes photographs of SH-5Y5Y cells infected with rVSV_(Ind)having different mutations in the M gene (panels C, D, E, F, I, J, K andL) or wild type M gene (panels A and G) and photographs of uninfectedSH-SY5S cells (panels B and H). Cells were incubated at 31° C. (panels Ato F) or 37° C. (panels G to L).

FIG. 9 includes photographs of BHK21 cells infected with rVSV_(NJ)having different mutations in the M gene (panels A, D, E, F, G, H, K, L,M and N) or wild type M gene (panels B and I), and photographs ofuninfected BHK21 cells (panels C and J). Cells were incubated at 31° C.(panels A to G) or 37° C. (panels H to N).

FIG. 10 includes photographs of SH-SY5Y cells infected with rVSV_(NJ)having different mutations in the M gene (panels A, D, E, F, G, H, K, L,M, and N) or having wild type M gene (panels B and I), and photographsof uninfected SH-SY5Y cells (panels C and J). Cells were incubated at31° C. (panels A to G) or 37° C. (panels H to N).

FIG. 11 includes graphs illustrating neurovirulence studies in SwissWebster mouse by intralateral ventricular injection: A) UV-irradiatedrVSV_(Ind) G21E/L111F/M51R, B) rVSV_(Ind) WT, C) rVSV_(Ind) M51R and D)rVSV_(Ind) G21E/L111F/M51R.

FIG. 12 includes graphs illustrating neurovirulence studies in SwissWebster mouse by intralateral ventricular injection: A) rVSV_(NJ) WT,B), rVSV_(NJ) M48R+M51R, C) rVSV_(NJ) G22E/M48R+M51R and D) rVSV_(NJ)G22E/L110F/M48R+M51R.

FIG. 13 (SEQ ID NOS: 11-15) A) illustrates the cloning of HIV-1 Gag-Engenes into the cDNA clone of rVSV. FIG. 13 B) is a Western blot analysisof BHK cells infected with rVSVs expressing Gag-En and having differentmutations to the M gene, and incubated at 31° C. FIG. 13 C) is a Westernblot analysis of BHK cells infected with rVSVs expressing Gag-En andhaving different mutations to the M gene, and incubated at 37° C.

FIG. 14 depicts a vaccination regime and a table of vaccination groups.

FIG. 15 depicts a graph of the frequency of peptide specific CD8+IFNgamma+ T cells among the different vaccination groups illustrated in theinsert stimulated with dimethyl sulfoxide (DMSO).

FIG. 16 is a graph illustrating the frequency of VSV N peptide specificCD8+IFN gamma+ T cells among the different vaccination groupsillustrated in the insert stimulated with VSV N.

FIG. 17 is a graph illustrating the frequency of HIV-1 Gag peptidespecific CD8+IFN gamma+ T cells among the different vaccination groupsillustrated in the insert stimulated with HIV-1 Gag.

FIG. 18 is a graph illustrating HIV-1 Gag specific antibody responsesamong the different vaccination groups illustrated in the insert.

FIG. 19 is a graph illustrating the frequency of VSV N peptide specificCD8+IFN gamma+ T cells among different vaccination groups vaccinatedwith various doses of rVSV of Table 1.

FIG. 20 is a graph illustrating the frequency of HIV-1 Gag peptidespecific CD8+IFN gamma+ T cells among different vaccination groupsvaccinated with various doses of rVSV of Table 1.

FIG. 21 is a graph illustrating HIV-1 Gag specific antibody responsesamong different vaccination groups vaccinated with various doses of rVSVof Table 1.

FIG. 22 (SEQ ID NOS: 16-26) illustrates the cloning of HIV-1 gag genelinked to nucleotides encoding human B cell and T cell epitopes of gp120and gp41 into the cDNA clone of rVSV_(Ind) G21E/L111F/M51R and rVSV_(NJ)G22E/M48R+M51R.

FIG. 23 (SEQ ID NOS: 11-15 and 27-31, respectively) illustrates thecloning of HIV-1 gag gene linked to nucleotides encoding human B celland T cell epitopes of gp41, nef, gp120, RT, Tat and Rev into the cDNAclone of rVSV_(Ind) G21E/L111F/M51R and rVSV_(NJ) G22E/M48R+M51R. Italso illustrates the cloning of HIV-1 pol and env genes into the cDNAclone of rVSV_(Ind) G21E/L111F/M51R and rVSV_(NJ) G22E/M48R+M51R.

FIG. 24 illustrates a Western blot analysis of BHK21 cells infected withrVSV_(Ind) G21E/L111F/M51R expressing Gag A, B, C, En, and RT andincubated at 31° C. and 37° C.

FIG. 25 illustrates a Western blot analysis of BHK21 cells infected withrVSV_(NJ) G22E/M48R+M51R expressing Gag A, B, C, En, and RT andincubated at 31° C. and 37° C.

FIG. 26 illustrates a Western blot analysis of BHK21 cells infected withrVSV_(Ind) G21E/L111F/M51R or rVSV_(NJ) G22E/M48R+M51R expressing HIV-1pol gene.

FIG. 27 illustrates a Western blot analysis of BHK21 cells infected withrVSV_(Ind) G21E/L111F/M51R or rVSV_(NJ) G22E/M48R+M51R expressing HIV-1gp160 mss gene.

FIG. 28 includes graphs illustrating VSV N and HIV-1 protein peptidespecific CD8+ T cell responses among the different vaccination groups ofTable 6: G1, G2, G3, G4, G5 and G5 without Env P18.

FIG. 29 is a graph illustrating VSV N and HIV-1 protein peptide specificCD8+ T cell responses in vaccination group G6 of Table 6. The amino acidsequences of VSV N peptide and peptides from HIV-1 proteins that areused to stimulated the splenocytes are shown (SEQ ID NOS: 32-37).

FIG. 30 illustrates the humoral immune responses against HIV-1 Gag(panel A) and Gp120 (panel B) induced in vaccination groups of Table 6,which were determined by Enzyme Linked Immunosorbant Assay (ELISA).

FIG. 31 depicts the HCV structural protein genes cloned intorVSV_(Ind)(GLM) and rVSV_(NJ)(GM) and rVSV_(NJ)(GLM).

FIG. 32 illustrates Western blot analyses of the expression of HCV Core(panel A), E1 (panel B), E2 (panel C), and NS4B (panel D) proteins fromthe rVSV_(Ind)(GLM), rVSV_(NJ)(GM), rVSV_(NJ)(GLM).

FIG. 33 depicts the HCV non-structural protein genes cloned intorVSV_(Ind)(GLM), rVSV_(NJ)(GM), and rVSV_(NJ)(GLM).

FIG. 34 Western blot analyses of the expression of HCV NS3 at 37° C. and31° C. (panel A), NS5A at 37° C. (panel B), and NS5B at 37° C. and 31°C. (panel C) from the rVSV_(Ind)(GLM).

FIG. 35 demonstrates the expression of HCV NS3, NS4B, NS5A, and NS5Bfrom rVSV_(NJ)(GM) at 37° C. (panel A) and the expression of HCV NS5ABat 37° C. (panel B). The protein expression was detected by Western blotanalysis.

FIG. 36 illustrates construction of a cDNA plasmid for the full lengthgenomic RNA of rVSV_(Ind)(GLM)-new.

FIG. 37 illustrates construction of a cDNA plasmid for the full lengthgenomic RNA of rVSV_(NJ)(GMM)-new.

FIG. 38 illustrates rVSV_(Ind)(GLM) with additional nucleotide changesshows the same temperature sensitivity as original rVSV_(Ind)(GLM)-new.

DETAILED DESCRIPTION OF THE INVENTION Overview

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Also, unless indicatedotherwise, except within the claims, the use of “or” includes “and” andvice versa. Non-limiting terms are not to be construed as limitingunless expressly stated or the context clearly indicates otherwise (forexample “including”, “having” and “comprising” typically indicate“including without limitation”). Singular forms including in the claimssuch as “a”, “an” and “the” include the plural reference unlessexpressly stated otherwise. “Consisting essentially of” means anyrecited elements are necessarily included, elements that wouldmaterially affect the basic and novel characteristics of the listedelements are excluded, and other elements may optionally be included.“Consisting of” means that all elements other than those listed areexcluded. Embodiments defined by each of these terms are within thescope of this invention. All publications cited and the prioritydocument are incorporated herein by reference.

All numerical designations, e.g., dimensions and weight, includingranges, are approximations that typically may be varied (+) or (−) byincrements of 0.1, 1.0, or 10.0, as appropriate. All numericaldesignations may be understood as preceded by the term “about”.

The term “administering” includes any method of delivery of a compoundof the present invention, including a pharmaceutical composition,vaccine or therapeutic agent, into a subject's system or to a particularregion in or on a subject. The phrases “systemic administration,”“administered systemically,” “peripheral administration” and“administered peripherally” as used herein mean the administration of acompound, drug or other material other than directly into the centralnervous system, such that it enters the patient's system and, thus, issubject to metabolism and other like processes, for example,subcutaneous administration. “Parenteral administration” and“administered parenterally” means modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intra-articular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

The term “amino acid” is known in the art. In general the abbreviationsused herein for designating the amino acids and the protective groupsare based on recommendations of the IUPAC-IUB Commission on BiochemicalNomenclature (see Biochemistry (1972) 11:1726-1732). For the amino acidsrelevant to the present invention the designations are: M: methionine,R: arginine, G: glycine, E: glutamic acid, L: leucine, F: phenylalanine.In certain embodiments, the amino acids used in the application of thisinvention are those naturally occurring amino acids found in proteins,or the naturally occurring anabolic or catabolic products of such aminoacids which contain amino and carboxyl groups. Particularly suitableamino acid side chains include side chains selected from those of thefollowing amino acids: glycine, alanine, valine, cysteine, leucine,isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid,glutamine, asparagine, lysine, arginine, proline, histidine,phenylalanine, tyrosine, and tryptophan.

The term “antibody” as used herein is intended to include wholeantibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), includingpolyclonal, monoclonal, recombinant and humanized antibodies andfragments thereof which specifically recognize and are able to bind anepitope of a protein. Antibodies can be fragmented using conventionaltechniques and the fragments screened for utility in the same manner.Thus, the term includes segments of proteolytically-cleaved orrecombinantly-prepared portions of an antibody molecule that are capableof selectively reacting with a certain protein. Nonlimiting examples ofsuch proteolytic and/or recombinant fragments include Fab, F(ab′)2,Fab′, Fv, and single chain antibodies (scFv) containing a V[L] and/orV[H] domain joined by a peptide linker. The scFvs may be covalently ornon-covalently linked to form antibodies having two or more bindingsites.

As used herein, the term “epitopes” refers to sites or fragments of apolypeptide or protein having antigenic or immunogenic activity in ananimal, preferably in a mammal. An epitope having immunogenic activityis a site or fragment of a polypeptide or protein that elicits an immuneresponse in an animal. An epitope having antigenic activity is a site orfragment of a polypeptide or protein to which an antibodyimmunospecifically binds as determined by any method well-known to oneof skill in the art, for example by immunoassays.

As used herein, the term “fragment” in the context of a proteinaceousagent refers to a peptide or polypeptide comprising an amino acidsequence of at least 2 contiguous amino acid residues, at least 5contiguous amino acid residues, at least 10 contiguous amino acidresidues, at least 15 contiguous amino acid residues, at least 20contiguous amino acid residues, at least 25 contiguous amino acidresidues, at least 40 contiguous amino acid residues, at least 50contiguous amino acid residues, at least 60 contiguous amino residues,at least 70 contiguous amino acid residues, at least 80 contiguous aminoacid residues, at least 90 contiguous amino acid residues, at least 100contiguous amino acid residues, at least 125 contiguous amino acidresidues, at least 150 contiguous amino acid residues, at least 175contiguous amino acid residues, at least 200 contiguous amino acidresidues, or at least 250 contiguous amino acid residues of the aminoacid sequence of a peptide, polypeptide or protein. In one embodiment, afragment of a full-length protein retains activity of the full-lengthprotein, e.g., immunogenic activity. In another embodiment, the fragmentof the full-length protein does not retain the activity of thefull-length protein, e.g., non-immunogenic activity.

As used herein, the term “fragment” in the context of a nucleic acidrefers to a nucleic acid comprising an nucleic acid sequence of at least2 contiguous nucleotides, at least 5 contiguous nucleotides, at least 10contiguous nucleotides, at least 15 contiguous nucleotides, at least 20contiguous nucleotides, at least 25 contiguous nucleotides, at least 30contiguous nucleotides, at least 35 contiguous nucleotides, at least 40contiguous nucleotides, at least 50 contiguous nucleotides, at least 60contiguous nucleotides, at least 70 contiguous nucleotides, at leastcontiguous 80 nucleotides, at least 90 contiguous nucleotides, at least100 contiguous nucleotides, at least 125 contiguous nucleotides, atleast 150 contiguous nucleotides, at least 175 contiguous nucleotides,at least 200 contiguous nucleotides, at least 250 contiguousnucleotides, at least 300 contiguous nucleotides, at least 350contiguous nucleotides, or at least 380 contiguous nucleotides of thenucleic acid sequence encoding a peptide, polypeptide or protein. In apreferred embodiment, a fragment of a nucleic acid encodes a peptide orpolypeptide that retains activity of the full-length protein, e.g.,immuogenic activity. In another embodiment, the fragment of thefull-length protein does not retain the activity of the full-lengthprotein, e.g., non-immunogenic activity.

The term “essentially noncytolytic” as used herein means that therecombinant vesicular stomatitis virus (rVSV) does not significantlydamage or kill the cells it infects.

The term “HIV” is known to one skilled in the art to refer to HumanImmunodeficiency Virus. There are two types of HIV: HIV-1 and HIV-2.There are many different strains of HIV-1. The strains of HIV-1 can beclassified into three groups: the “major” group M, the “outlier” group Oand the “new” group N. These three groups may represent three separateintroductions of simian immunodeficiency virus into humans. Within theM-group there are at least ten subtypes or clades: e.g., clade A, B, C,D, E, F, G, H, I, J, and K. A “clade” is a group of organisms, such as aspecies, whose members share homologous features derived from a commonancestor. Any reference to HIV-1 in this application includes all ofthese strains.

The term “non-infectious” means of reduced to non-existent ability toinfect.

As used herein, the terms “subject” or “patient” are usedinterchangeably. As used herein, the terms “subject” and “subjects”refers to either a human or non-human animal.

The term “pharmaceutical delivery device” refers to any device that maybe used to administer a therapeutic agent or agents to a subject.Non-limiting examples of pharmaceutical delivery devices includehypodermic syringes, multichamber syringes, stents, catheters,transcutaneous patches, microneedles, microabraders, and implantablecontrolled release devices. In one embodiment, the term “pharmaceuticaldelivery device” refers to a dual-chambered syringe capable of mixingtwo compounds prior to injection.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

The terms “polynucleotide”, “nucleic acid sequence” and “nucleic acid”are used interchangeably to refer to a polymeric form of nucleotides ofany length, either deoxyribonucleotides or ribonucleotides, or analogsthereof. The following are non-limiting examples of polynucleotides:coding or non-coding regions of a gene or gene fragment, loci (locus)defined from linkage analysis, exons, introns, messenger RNA (mRNA),transfer RNA (tRNA), ribosomal RNA (rRNA), ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.The term “recombinant” polynucleotide means a polynucleotide of genomic,cDNA, semi-synthetic, or synthetic origin, which either does not occurin nature or is linked to another polynucleotide in a non-naturalarrangement. An “oligonucleotide” refers to a single strandedpolynucleotide having less than about 100 nucleotides, less than about,e.g., 75, 50, 25, or 10 nucleotides.

The terms “polypeptide”, “peptide” and “protein” (if single chain) areused interchangeably herein to refer to polymers of amino acids. Thepolymer may be linear or branched, it may comprise modified amino acids,and it may be interrupted by non-amino acids. The terms also encompassan amino acid polymer that has been modified; for example, disulfidebond formation, glycosylation, lipidation, acetylation, phosphorylation,or any other manipulation, such as conjugation with a labelingcomponent.

In certain embodiments, polypeptides of the invention may be synthesizedchemically, ribosomally in a cell free system, or ribosomally within acell. Chemical synthesis of polypeptides of the invention may be carriedout using a variety of art recognized methods, including stepwise solidphase synthesis, semi-synthesis through the conformationally-assistedre-ligation of peptide fragments, enzymatic ligation of cloned orsynthetic peptide segments, and chemical ligation. Native chemicalligation employs a chemoselective reaction of two unprotected peptidesegments to produce a transient thioester-linked intermediate. Thetransient thioester-linked intermediate then spontaneously undergoes arearrangement to provide the full length ligation product having anative peptide bond at the ligation site. Full length ligation productsare chemically identical to proteins produced by cell free synthesis.Full length ligation products may be refolded and/or oxidized, asallowed, to form native disulfide-containing protein molecules (seee.g., U.S. Pat. Nos. 6,184,344 and 6,174,530; and T. W. Muir et al.,Curr. Opin. Biotech. (1993): vol. 4, p 420; M. Miller, et al., Science(1989): vol. 246, p 1149; A. Wlodawer, et al., Science (1989): vol. 245,p 616; L. H. Huang, et al., Biochemistry (1991): vol. 30, p 7402; M.Schnolzer, et al., Int. J. Pept. Prot. Res. (1992): vol. 40, p 180-193;K. Rajarathnam, et al., Science (1994): vol. 264, p 90; R. E. Offord,“Chemical Approaches to Protein Engineering”, in Protein Design and theDevelopment of New therapeutics and Vaccines, J. B. Hook, G. Poste,Eds., (Plenum Press, New York, 1990) pp. 253-282; C. J. A. Wallace, etal., J. Biol. Chem. (1992): vol. 267, p 3852; L. Abrahmsen, et al.,Biochemistry (1991): vol. 30, p 4151; T. K. Chang, et al., Proc. Natl.Acad. Sci. USA (1994) 91: 12544-12548; M. Schnlzer, et al., Science(1992): vol., 3256, p 221; and K. Akaji, et al., Chem. Pharm. Bull.(Tokyo) (1985) 33: 184).

“VSV” is used to refer to vesicular stomatitis virus.

“rVSV” is used to refer to a recombinant vesicular stomatitis virus.

The term “Indiana”, and “IND” are used to refer to the VSV serotypeIndiana (VSV_(Ind)).

The term “New Jersey”, and “NJ” are used to refer to the VSV serotypeNew Jersey (VSV_(NJ)).

“MWT” “M(WT)” are used to refer to a wild type M protein. The nucleotidesequence of wild type M gene of the VSV_(Ind) may comprise thenucleotide sequence represented by SEQ ID NO: 1. The amino acid sequenceof wild type M protein of the VSV_(Ind) may comprise the amino acidsequence represented by SEQ ID NO: 3. The nucleotide sequence of wildtype M gene of the VSV_(NJ) may comprise the nucleotide sequencerepresented by SEQ ID NO: 5. The amino acid sequence of wild type Mprotein of the VSV_(NJ) may comprise the amino acid sequence representedby SEQ ID NO: 8. “M51R” is used to refer to an M(WT) in the VSV_(Ind)having a methionine changed to an arginine at position 51. “G21E” isused to refer to an M(WT) in VSV_(Ind) having a glycine changed to aglutamic acid at position 21. “L111A” is used to refer to an M(WT) inVSV_(Ind) having a leucine (L) changed to alanine (A) at position 111.“G22E” is used to refer to an M(WT) in VSV_(NJ) having a glycine (G)changed to glutamic acid (E) at position 22. “L110A” is used to refer toan M(WT) in VSV_(NJ) having a leucine (L) changed to alanine (A) atposition 110. “M48R+M51R” or “M48R/M51R” is used to refer to an M(WT) inVSV_(NJ) having a methionine (M) changed to an arginine (R) at positions48 and 51. “rVSV_(Ind) M(G21E/L111A/M51R)” or “rVSV_(Ind) (GLM)-new” areused to refer to a rVSV_(Ind) having an M(WT) having a glycine changedto a glutamic acid at position 21, a leucine changed to alanine atposition 111 and a methionine changed to an arginine at position 51.“rVSV_(NJ) M(G22E/M48R/M51R)” “rVSV_(NJ) M(G22E/M48R+M51R)” or“rVSV_(NJ) (GM)” are used to refer to rVSV_(NJ) having an M(WT) having aglycine changed to a glutamic acid at position 22 and a methioninechanged to an arginine at positions 48 and 51. “rVSV_(NJ)(GM)-new”refers to rVSV_(NJ)(GM) with the codons of the present invention.“rVSV_(NJ) M(G22E/L110F/M48R/M51R)” or “rVSV_(NJ)M(G22E/L110F/M48R+M51R)” or “rVSV_(NJ) (GLM)” are used to refer torVSV_(NJ) having an M(WT) having a glycine changed to a glutamic acid atposition 22, a leucine changed to a phenylalanine at position 110 and amethionine changed to an arginine at positions 48 and 51. “rVSV_(NJ) M(G22E/L110A/M48R/M51R)”, “rVSV_(NJ) (G22E/L110A/M48R+M51R)” or“rVSV_(NJ) (GLM)-new” is used to refer to rVSV_(NJ) having an M(WT)having a glycine changed to a glutamic acid at position 22, a leucinechanged to alanine at position 110 and a methionine changed to anarginine at positions 48 and 51.

Overview

The inventors generated novel M proteins and novel attenuated rVSVscapable of producing the novel M proteins. The novel proteins of thepresent invention may include: M(G21E/L111A/M51R), andM(G22E/M48R/M51R). The novel attenuated rVSVs of the present inventionmay be used as protein expression and vaccine vectors and in methods forpreventing or treating infections. The rVSV of the present invention maybe applied to make vaccines for the infectious diseases of human andother animals to induce cellular and humoral immune responses.

Isolated Proteins and M Proteins

In one embodiment, the present invention relates to isolated proteinsand to nucleotide sequences that encode the isolated proteins. As such,in one embodiment the present invention relates to an isolated peptidecomprising an amino acid sequence selected from amino acid sequenceslisted as SEQ ID NOs: 4 and 9. In another embodiment, the presentinvention relates to isolated nucleotide sequences comprising anucleotide sequence selected from the polynucleotides listed as SEQ IDNOs: 2 and 6. The isolated peptides and nucleotide sequences may beprovided in purified form. The nucleotides and amino acid sequences ofthe present invention may be artificially made or synthesized by knownmethods in the art.

In one embodiment, the present invention relates to novel VSV M proteinshaving at least one of the following substitutions: M(G21E/L111A/M51R),M(G22E/M48R/M51R) or M(G22E/L110A/M48R/M51R).

The E at positions 21 or 22 as the case may be, is encoded by the codongaa and R at positions 48 and 51, as the case may be, is encoded bycodon cga. The A at position 111 may preferably be encoded by the codongca.

In one aspect the present invention relates to a VSV M proteincomprising an amino acid sequence selected from the amino acid sequenceslisted as SEQ ID NOs: 4, 9 and 10. In another embodiment the presentinvention relates to nucleotide sequences which encode for the novel VSVM proteins of the present invention. The nucleotide sequences may beselected from the group of sequences listed as SEQ ID NOs: 2, 6 and 7.

Methods of Preventing or Treating an Infection

Provided are methods of inducing an immune response, preventing ortreating infections. In one embodiment, the methods may includeadministering to a subject: (a) an effective amount of a vaccinecomprising an attenuated rVSV of one serotype having (i) a firstmodified M protein, the first modified M protein comprising the aminoacid sequence of SEQ ID NO: 3 including the following substitutions:G21E/L111A/M51R, and (ii) an epitope of the pathogen; and (b) aneffective amount of another vaccine comprising an attenuated rVSV ofanother serotype having: (i) a second modified M protein, the secondmodified M protein comprising the amino acid sequence of SEQ ID NO: 8including the following substitutions: G22E/M48R/M51R, and (ii) theepitope of the pathogen. In embodiments of the present invention, themethods may include administering to a subject (a) an effective amountof rVSV_(Ind) M(G21E/L111A/M51R), and (b) an effective amount ofrVSV_(NJ) M(G22E/M48R/M51R) or M(G22E/L110A/M48R/M51R) in a prime-boostimmunization modality. The E at positions 21 or 22 as the case may be,is encoded by the codon gaa and R at positions 48 and 51, as the casemay be, is encoded by codon cga. The A at position 111 or 110 maypreferably be encoded by the codon gca.

The term “effective amount” as used herein means an amount effective andat dosages and for periods of time necessary to achieve the desiredresult.

In certain embodiments, (a) is administered to the subject before (b) isadministered to the subject.

In certain embodiments, (b) is administered to the subject more than onetime over the course of treating or preventing.

In certain embodiments, (a) is administered to the subject in needthereof and (b) is administered to the subject in need thereof at aboutweeks three, eight and sixteen post-administration of (a).

In certain embodiments, (b) is administered to the subject before (a) isadministered to the subject.

In certain embodiments, (a) is administered to the subject more than onetime over the course of treating or preventing.

In certain embodiments, (b) is administered to the subject in needthereof and (a) is administered to the subject in need thereof at aboutweeks three, eight and sixteen post-administration of (b).

Recombinant Virus

In certain embodiments, present invention relates to a recombinantvesicular stomatitis virus (rVSV) which may be a full length VSV,essentially non-cytolytic, avirulent, capable of inducing an immuneresponse in a subject, capable of reproducing virus particles to a hightire at permissive temperatures, reproducing virus particles to a lowtitre at semi-permissive temperatures and which may be incapable ofproducing virus at non-permissive temperatures, and that can express anepitope of a foreign pathogen. The rVSV of the present invention may becapable of inducing humoral, cellular and mucosal immune responses.

In one embodiment, the present invention relates to rVSV_(Ind) andrVSV_(NJ). The rVSV_(Ind) may be a full length, essentially noncytolyticrVSV_(Ind) M(G21E/L111A/M51R) capable of producing virus particles at apermissible temperature of about 31° C., and which may be incapable ofor poorly capable of producing virus particles at a semi-permissivetemperatures of about 37° C. and incapable of producing virus particlesat non-permissive temperatures above 37° C., for example 39° C. Incertain embodiments, the rVSV_(Ind) may include a M(G21E/L111A/M51R). Incertain embodiments, the rVSV_(Ind) may include an M gene comprising anucleotide sequence SEQ ID NO: 2.

In certain embodiments, the rVSV is a full-length, essentiallynoncytolytic rVSV_(NJ) M (G22E/M48R/M51R) or M(G22/L110A/M48R/M51R). Incertain embodiments, the rVSV is an essentially noncytolytic rVSV_(NJ)including an M gene, wherein the nucleotide sequence of the M gene isselected from SEQ ID NO: 6 and SEQ ID NO: 7.

The rVSVs of the present invention can be prepared using techniquesknown in the art. In one embodiment, the rVSVs may be introduced in ahost cell under conditions suitable for the replication and expressionof the rVSV in the host. Accordingly, the present invention alsoprovides a cell having a rVSV_(Ind) wherein the amino acid sequence ofthe virus' M protein is modified to provide an essentially non-cytotoxicwhich also allows the rVSV_(Ind) to effectively replicate at permissibletemperature but may not replicate at non-permissible temperature.

As such, the present invention relates also to a cell having one or moreof the recombinant VSVs of the present invention.

Vaccines or Immunogenic Compositions of the Invention

The present invention further features vaccines or immunogeniccompositions comprising one or more of the rVSVs of the presentinvention. In one embodiment, the present invention features vaccines orimmunogenic compositions comprising an rVSV_(Ind) and vaccines orimmunogenic compositions comprising an rVSV_(NJ), as described above.

In one embodiment, the vaccines may include rVSVs expressing an epitopeof a pathogen. In another embodiment, the vaccines may include a mixtureor cocktail of rVSVs expressing different epitopes of a pathogen (seeTable 6, vaccination groups 5 and 6).

The vaccine or immunogenic compositions of the invention are suitablefor administration to subjects in a biologically compatible form invivo. The expression “biologically compatible form suitable foradministration in vivo” as used herein means a form of the substance tobe administered in which any toxic effects are outweighed by thetherapeutic effects. The substances maybe administered to any animal orsubject, preferably humans. The vaccines of the present invention may beprovided as a lyophilized preparation. The vaccines of the presentinvention may also be provided as a solution that can be frozen fortransportation. Additionally, the vaccines may contain suitablepreservatives such as glycerol or may be formulated withoutpreservatives. If appropriate (i.e. no damage to the VSV in thevaccine), the vaccines may also contain suitable diluents, adjuvantsand/or carriers.

The dose of the vaccine may vary according to factors such as thedisease state, age, sex, and weight of the individual, and the abilityof antibody to elicit a desired response in the individual. Dosageregime may be adjusted to provide the optimum therapeutic response. Thedose of the vaccine may also be varied to provide optimum preventativedose response depending upon the circumstances.

Kits

The present invention provides kits, for example for preventing ortreating an infection. For example, a kit may comprise one or morepharmaceutical compositions or vaccines as described above andoptionally instructions for their use. In still other embodiments, theinvention provides kits comprising one or more pharmaceuticalcompositions or vaccines and one or more devices for accomplishingadministration of such compositions.

Kit components may be packaged for either manual or partially or whollyautomated practice of the foregoing methods. In other embodimentsinvolving kits, this invention contemplates a kit including compositionsof the present invention, and optionally instructions for their use.Such kits may have a variety of uses, including, for example, imaging,diagnosis, therapy, and other applications.

Advantages and Unique Features of the rVSVs of the Present Invention

Novel and Unusual Features of the Invention:

Normal assembly and release of rVSV_(Ind) M(G21E/L111A/M51) atpermissive temperature (about 31° C.) made it possible to amplify thenew mutant rVSVs at the permissible temperature to a high titre to makeviral stock. The assembly defectiveness of rVSV_(Ind) M(G21E/L111A/M51)at non-permissive temperature (about 37° C. (around body temperature)increased the safety of the using the rVSV in human and other animals bysignificantly reducing the number of progeny infectious viruses at thenon-permissive temperature. The addition of these mutations to thepre-existing M51R mutation in the M protein of rVSV_(Ind) furtherattenuated the virulence of VSV_(Ind).

Prior to the present invention it was unknown that including thesubstitution L111A would result in rVSV having normal assembly atpermissive temperatures and assembly defectiveness at non-permissivetemperatures. The advantage of having a L111A substitution instead ofL111F is that the former has less chance of reverting back to a wildtype form.

The E at positions 21, which may be encoded by the codon gaa and R atpositions 48 and 51, as the case may be, being encoded by codon cgadecreases the chances of the mutants reverting back to wild type.

Previously the inventors developed attenuated rVSV_(Ind) with themutations of G21E, M51R, and L111F in the M gene. The amino acid changeswere accomplished by changing nucleotides. Nucleotide codon for Glycine21, GGG was changed to GAA for Glutamic acid with 2 nucleotide changes.Nucleotide codon for Methionine51, ATG was changed to AGG for Argininewith 1 nucleotide change. Nucleotide codon for Leucine, TTG was changedto TTT for Phenylalanine with 1 nucleotide change. The initialnucleotide changes to mutate M51 to R and L111 to F were only one ineach amino acid. However, it is very likely that, if we change morenucleotides to change amino acids, will diminish the chances of thechanged amino acids reverting back to the original amino acids.Therefore, the iventors mutated AGG of M51R to CGA to have thenucleotide codon changed completely in all 3 nucleotides. TTT ofPhenylalanine was mutated to GCA of Alanine to have 3 nucleotides werecompletely changed. The resulting mutations in the M protein ofrVSV_(Ind) (GLM)-New are G21E/L111A/M51R with nucleotide changes ofGAA(E)/GCA(A)/CGA(R). More nucleotides changes in a codon for a aminoacid change will increase the stability of the temperature sensitivemutations in the rVSV_(Ind)(GLM). Likewise more nucleotides for M48R andM51R in the rVSV_(NJ)(GMM) mutant. The initial changes in the nucleotidecodon for the mutations G22E/M48R/M51R were GAA(E)/AGG(R)/AGG(R). Thesenucleotide codons were further changed to GAA(E)/CGA(R)/CGA(R) to haveall three nucleotides for the Arginine changed.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitation.

EXAMPLES

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way.

Example 1 Introduction of Mutations into the M Genes of rVSV_(Ind) andrVSV_(NJ) and Recovery of Recombinant VSV by Reverse Genetics

Mutations were introduced into the M gene of VSV_(Ind) (FIG. 1) andVSV_(NJ) (FIG. 2). Nucleotide sequences encoding the amino acids at eachposition were mutated by the mega-primer PCR method. Each mutation isexpressed as a substitution of an amino acid at a specific position(e.g., M51 in M51R) with another amino acid (e.g., R in M51R). In orderto attenuate further the virulence of VSV, the inventors combinedmutations (G21E and L11F) in the tsO23 with methionine to argininemutations (M51R) in the M gene, which reduced inhibitory activity of Mprotein on the cellular protein synthesis, in addition, reduced theassembly of the VSV particles at non-permissive (39° C.) andsemi-permissive (37° C.) temperatures.

Wild type and mutant recombinant vesicular stomatitis viruses (rVSV)were recovered from the cDNA plasmids by reverse genetics (FIG. 3). TheVSV reverse genetics employs the BHK21 cells expressing DNA dependantRNA polymerase of bacteriophage T7 (T7) and a plasmid which encodes fulllength genomic RNA of VSV (pVSV) and 3 plasmids expressing nucleocapsidprotein (pN), phosphoprotein (pP), and VSV polymerase L protein (pL).The transcription of the full length genomic RNA and the messenger RNAsfor N, P, and L proteins are under the control of T7 RNA polymerase.Internal ribosome entry site (IRES) at the upstream of each VSV N, P,and L gene enhances the translation of proteins. The plasmids aretransfected into BHK-T7 cells with Lipofectamine™ 2000 in concentrationsof 10 μg of pN, 10 μg of pPμ5 g of pL, and 15 μg of pVSV. The culturemedia from the transfected cells were harvested when the cells showedabout 50-70% of CPE.

Example 2 The Mutation, L111F in the M Gene Significantly Reduces theBurst Sizes of rVSV_(Ind) at Semi-Permissive Temperature, 37° C.

The recovered viruses were purified 3 times by plaque picking and wereamplified for a larger volume of stock viruses by infecting BHK21 cellswith an MOI of 0.1 at 31° C. The inventors infected BHK21 cells andhuman neuroblastoma cells, SH-SY5Y with an MOI of 3 of rVSVs. Theinfected cells were incubated at permissive temperature (31° C.) andsemi-permissive temperature (37° C., body temperature) to determine thetemperature sensitivity of the new M mutants in the assembly of virusparticles. Culture media from the infected cells were collected every 2hours until 10 hours after the infection, and the number of infectiousviral particles in the culture media was determined by plaque assay withVero E6 cells. The cells infected with the mutant viruses for the plaqueassay were incubated at 31° C. Wild type and all mutants replicatedequally well and produced similar titre of infectious viruses all alongthe period of 10 hrs of infection (FIGS. 4A and C). However, the mutantsof rVSV_(Ind), rVSV_(Ind)-G21E/L111F and rVSV_(Ind)-G21E/L111F/M51Rreplicated in significantly lower titre than wild type or M51R mutant ofrVSV_(Ind) at 37° C. The differences in producing infectious particlesbetween the wild type and the new M mutant, rVSV_(Ind)-G21E/L111F/M51Rwere as large as four logs (FIGS. 4B and D). The error bars in FIG. 4represent standard error of the mean. The P values for viral titers ofrVSV_(Ind)-G21E/L111F/M51R at 4 hrs, 6 hrs, 8 hrs, and 10 hrspostinfection obtained by comparing them to titers of rVSV_(Ind)-WT arep<0.0001, p=0.0055, p=0.0040, and p=0.0015 in FIG. 4B. The P values forviral titers of rVSV_(Ind)-G21E/L111F at 4 hrs, 6 hrs, 8 hrs, and 10 hrspostinfection obtained by comparing them to titers of rVSV_(Ind)-WT arep<0.0001, p=0.0055, p=0.0041, and p=0.0016 in FIG. 4B. The P values inFIG. 4D were <0.005. P values were computed by using two-sidedindependent t tests.

Example 3 Mutations in the M Gene of rVSV_(NJ), G22E and L110F do notReduce the Burst Size of the rVSV_(NJ) at 37° C.

BHK21 cells were infected with an MOI of 3 of rVSV_(NJ), wild type and Mgene mutants, incubated at both 37° C. (semi-permissive temperature) and31° C. (permissive temperature) and the culture media was harvested at10 hrs post infection. The viral titer of each virus in the culturemedia was determined by plaque assay using Vero E6 cells. The averageviral titre from the duplicate samples is shown in the table. Wild typeand all M mutants of rVSV_(NJ) replicated equally well at bothtemperatures 31° C. and 37° C. (FIG. 5), indicating the introduction ofG22E and L110F mutations into the M gene of rVSV_(NJ) did not affect theassembly and release of the virus at 37° C. The error bars in FIG. 5represent standard error of the mean.

Example 4 Assembly Defectiveness at Semi-Permissive Temperature by L111FMutation in the M Gene of rVSV_(Ind) does not Affect the Expression ofVSV Proteins

BHK21 cells were infected with an MOI of 6 of rVSV_(Ind) and rVSV_(NJ),wild type and M gene mutants. The infected cells were incubated at both37° C. and 31° C. for 6 hrs. The infected cells were lysed at 6 hrspost-infection, 5 μg of total protein was loaded to the SDS-PAGE gel,and rVSV proteins were detected by Western blot analysis using rabbitantiserum against VSV_(Ind) and VSV_(NJ) (1:5000 dilution). The resultdemonstrate that in spite of the mutations that reduced the burst sizeof the rVSV_(Ind) at 37° C. (L111F in G21E/L111F and G21E/L111F/M51R),the level of VSV protein expression is comparable to the wild typerVSV_(Ind) (FIG. 6A). Wild type and all M mutants of rVSV_(NJ) expressedtheir proteins in a similar level at both 31° C. and 37° C. (FIG. 6B).

Example 5 Combined Mutations, G21E/L111F/M51R in the M Gene ofrVSV_(Ind) Reduced Cytopathogenicity Significantly in BHK21 Cells andHuman Neuroblastoma Cells, SH-SY5Y

In order to examine the effects of the mutations, L111F and M51R of Mgene of rVSV_(Ind) on the cytopathogenicity, the inventors infectedBHK21 cells (FIG. 7) and human neuroblastoma cells (FIG. 8) with an MOIof 0.1 of rVSV_(Ind). The typical cytopathic effects by the VSV arerounding-up of infected cells and cell lysis. At 20 hrs after infection,the cytopathic effect caused by the rVSV_(Ind)-G21E/L111F/M51R wascompared with those by the other rVSV_(Ind). In 20 hrs of infection withthe rVSV_(Ind)-G21E/L111F/M51R mutant showed the most reduced cytopathiceffects, none or small number of round-up cells at 37° C. and thecombination of the mutations L111F and M51R (FIG. 7N and FIG. 8L)further attenuated the cytopathogenicity of the virus than the singlemutation of each in both BHK21 and SH-SY5Y cells.

Example 6 Reduced Cytopathic Effects in the BHK21 Cell by the rVSV_(NJ)with the M Gene Mutations, G22E/M48R+M51R and G22E/L110F/M48R+M51R

In order to examine the effects of the mutations, G22E, L110F andM48R+M51R of M gene of rVSV_(NJ) on the cytopathogenicity, the inventorsinfected BHK21 cells and human neuroblastoma cells with an MOI of 0.1 ofrVSV_(NJ). At 20 hrs after infection, the inventors compared thecytopathic effects (cell round-up and lysis) caused by the rVSV_(NJ)wild type and other M mutants. In 20 hrs of infection with theG22E/M48R+M51R mutant and G22E/L110F/M48R+M51R mutant showed the leastcytopathic effects at 37° C. and the combination of the mutations, G22Eand M48R+M51R further attenuated the cytopathogenicity of the rVSV_(NJ)in both BHK21 (FIGS. 9L and 9N) and SH-SY5Y cells (FIGS. 10L and 10N).

Example 7 rVSV_(Ind) with the Mutations of G21E/L111F/M51R and rVSV_(NJ)with the Mutations of G22E/M48R+M51R and G22E/L110F/M48R+M51R in the MProtein was Attenuated to a Degree that Mice Injected with the Virusinto the Brain Showed No Neurological Signs or any Symptoms Such asWeight Loss

VSV does not show the neurotropism in host animal during the naturalinfection through the skin abrasion or sandfly or mosquito bites.Nevertheless, when wild type VSV is injected directly into the nose orbrain in mice or monkeys, the animals demonstrate neurological symptoms.In order to examine the neurovirulence of the new M mutants of VSVs, theinventors injected Swiss Webster mice with 1×10⁶ PFU of mutant VSV and1×10³ PFU of wild type VSV into the intralateral ventricle of the brain.The inventors purchased 5-week-old Swiss Webster mice with intralateralventricular implant from Charles River laboratory. Three mice/group wereinjected with viruses after one week of arrival to the animal facility.After the viral injection, mice were observed for neurological signs andwere weighed every two days for 4 weeks. The mice injected with 1×10³PFU of wild type rVSV_(Ind) died within four days after injection (FIG.11B). Two mice injected with 1×10⁶ PFU of rVSV_(Ind)-M51R (FIG. 11C)lost about 20% of body weights 6 days after injection and the bodyweight bounced back to normal weight at about 14 days after injection.One mice injected with the rVSV_(Ind)-M51R did not lose the body weightthrough the experiment. All three mice injected withrVSV_(Ind)-G21E/L111F/M51R showed no sign of illness and did not losetheir body weight for 4 weeks until the mice were sacrificed (FIG. 11D)indicating the combination of mutations, G21E/L111F and M51Rsignificantly attenuated the rVSV_(Ind) and lost its neurovirulence inmice. One mice injected with rVSV_(NJ)-WT showed paralysis in both hindlegs and it was sacrificed on day 6 after injection (FIG. 12A). Twoother mice injected with rVSV_(NJ)-WT and mice injected withrVSV_(NJ)-M48R+M51R, rVSV_(NJ)-G22E/M48R+M51R, orrVSV_(NJ)-G22E/L110F/M48R+M51R showed no sign of illness and weight lossduring 4 weeks after injection (FIGS. 12B, 12C, and 12D). The error barsin FIGS. 11 and 12 represent standard error of the mean.

Example 8 Generation of rVSVs Expressing HIV-1 Gag Protein as a Gene ofInterest; HIV-1 Gag Proteins are Expressed Equally Well at Both 31° C.and 37° C.

The newly generated M mutants, G21E/L111F/M51R mutant of rVSV_(Ind) andG22E/M48R+M51R and G22E/L110F/M48R+M51R mutants of rVSV_(NJ)demonstrated the reduced cytopathogenicity and comparable proteinexpressions to the wild type rVSV at 37° C. In order to be used as avaccine vector, the new M mutants of rVSV should induce good immuneresponses in vivo, both humoral and cellular immune responses. When itis expressed alone in cells, HIV-1 gag proteins produce virus likeparticles and the virus like particles are secreted from the cells.Therefore, the gag protein was suitable protein to express from the newM mutants of rVSV to examine both cellular and humoral immune response.In addition, the CD8+ cytotoxic T cell epitope, H-2K^(d)-restrictedpeptide (NH₂-AMQMLKETI-COOH) (SEQ ID NO: 33) in the HIV-1 Gag is wellstudied in the Balb/c mouse. The inventors inserted the full-lengthHIV-1 gag gene linked to conserved human CD8+ T cell epitopes from gp41,gp120, and nef protein of HIV-1 (Gag-En). The Gag-En gene was insertedinto the junction of G gene and L gene in the full-length cDNA clones ofwild type (WT) and G21E/L111F/M51R (GLM) of rVSV_(Ind) and wild type,G22E/M48R+M51R (GM), and G22E/L110F/M48R+M51R(GLM) of rVSV_(NJ). TherVSVs were recovered from the cDNA clones by reverse genetics asdescribed in FIG. 3 and the expression of Gag-En from the rVSVs wasexamined by Western blot analysis using monoclonal antibody againstHIV-1 p24. For the Western blot analysis, BHK21 cells were infected withMOI of 6 of rVSVs and incubated at 31° C. and 37° C., and cell lysateswere prepared at 6 hrs post-infection. Total protein of 5 μg was loadedonto the SDS-PAGE for the Western blot analysis. rVSV_(Ind)(GLM)-Gag-En,rVSV_(NJ)(GM)-Gag-En, and rVSV_(NJ)(GLM)-Gag-En expressed Gag-En protein(64 kDa) well at semi-permissive temperature (37° C., FIG. 13C) and theexpression level was similar to that at permissive temperature (31° C.,FIG. 13B).

Example 9 Vaccination Regimen with rVSV in Mice

Six Balb/c mice per group were vaccinated with the prime-boost regimenillustrated in FIG. 14. Mice were grouped according to the vaccinevector type (wild type vs. mutant) and regimen, e. g., priming andboosting with the same serotype of rVSV or alternating the two serotypesfor priming and boost. The mice were prime-vaccinated intramuscularlywith 5×10⁶ PFU of rVSVs at age of 6 weeks. Three weeks after thepriming, mice were boost-vaccinated with the same dose of rVSV as theprime vaccination. A week after the booster injection, splenocytes andsera were collected to detect the HIV-1 Gag specific CD8+ T cell immuneresponses and humoral immune responses.

Example 10 Priming with rVSV_(Ind)(GLM)-HIV-1 Gag-En and Booster withrVSV_(NJ)(GLM)-HIV-1 Gag-En Induces Strongest CD8+ T Cell ImmuneResponses Against HIV-1 Gag

The T cells stimulated by the interaction with MHC I molecule on theantigen presenting cells loaded with the peptide enhances the secretionof interferon-γ (IFN-γ), which indicates the antigen specific T cellimmune responses. The splenocytes were double stained with FITC-anti-CD8and APC-anti-IFN-γ for CD8+ T cells with the increased intracellularINF-γ. In order to examine the HIV-1 Gag protein specific CD8+ T cellimmune response, splenocytes were isolated and 1×10⁶ cells werestimulated with H-2K^(d)-restricted HIV-1 Gag peptide,NH₂-AMQMLKETI-COOH (SEQ ID NO: 33), the cells were stained with FITC ratanti-mouse CD8 for the CD8 molecules and stained with APC rat anti-mouseIFN-γ for intracellular IFN-γ. The secretion of IFN-γ was blocked withBrefeldin A before staining them. VSV specific CD8+ T cell immuneresponses were examined with the use of nucleocapsid specific peptide,IN275: NH2-MPYLIDFGL-COOH (SEQ ID NO: 32). Peptide specific CD8+-IFN-γ+T cells were counted with FACSCalibur, a flowcytometer. The splenocytestreated with DMSO (solvent for the peptide, FIG. 15) did not stimulatethe CD8+ T cells indicating that treating the splenocytes with the VSV Npeptides and HIV-1 Gag peptides stimulated specifically CD8+ T cells inFIG. 16 and FIG. 17. Prime and boost immunization by alternating twoserotypes of rVSV(WT) or rVSV(GLM) mutants induced stronger CD8+ T cellimmune responses against VSV as well as HIV-1 Gag proteins than using asingle serotype of rVSV for the prime and boost vaccinations as seen ingroups 1, 2, 5, 6, 9, and 10. (FIG. 16 and FIG. 17). With thevaccination with the new M mutants or rVSV, CD8+ T cell responsesagainst HIV-1 Gag protein was induced better when mice were vaccinatedwith rVSV_(Ind)(GLM)-Gag for priming and rVSV_(NJ)(GLM)-Gag for boostingthan vice versa (FIG. 17, group 6 vs. group 10). P value on the group 6in FIG. 17 was computed by using two-sided independent sample t test,and the result was compared to that for the group 10. The error bars inFIGS. 16 and 17 represent standard error of the mean.

Example 11 HIV-1 Gag Specific Humoral Immune Responses

Generation of HIV-1 Gag specific antibody was examined with the serumcollected at a week after the boost immunization. The Gag specificantibody titer was determined by the indirect enzyme-linkedimmunosorbent assay (ELISA). For the ELISA, 96 well ELISA plate wascoated with recombinant p55 at a concentration of 125 ng/well. The mouseserum was diluted 1:100. The antibody bound to the antigen, p55 wasdetected with secondary antibody, sheep anti-mouse IgG-HRP. Theenzymatic activity of HRP was detected by adding substrates, a mixtureof hydrogen peroxide and tetramethylbenzidine. The OD of each sample wasread at the wavelength of 450 with the microplate reader. The humoralimmune responses against HIV-1 Gag (generation of antibody against HIV-1Gag) was induced well in mice vaccinated with the new M mutants, and thebest humoral immune responses against HIV-1 Gag were induced when twoserotypes of rVSV(WT) or rVSV(GLM) were alternated for prime and boosterinjection (FIG. 18, groups 5, 6, 9, and 10). As for the utilization ofthe new M mutants for inducing humoral immune responses against foreignproteins, priming with rVSV_(Ind)(GLM)-Gag and booster withrVSV_(NJ)(GLM)-Gag worked better than vice versa (Gig. 18, group 6 vs.group 10). The error bars in FIG. 18 represent standard error of themean.

Example 12 Increasing Doses of rVSV_(Ind)(GLM), rVSV_(NJ)(GM), andrVSV_(NJ)(GLM) for Vaccination Induced Stronger Immune Responses in Mice

As illustrated in FIG. 17 and FIG. 18, stronger HIV-1 Gag specific CD8+T cell immune responses and humoral immune responses were induced whenmice were prime vaccinated with rVSV_(Ind)(GLM)-Gag and boostervaccinated with rVSV_(NJ)(GLM)-Gag. However the immune responses werenot as strong as those induced with rVSV_(Ind)(WT)-Gag andrVSV_(NJ)(WT)-Gag. Therefore, the inventors wanted to examine whetherthey could increase the immune responses, if they increase the doses ofthe rVSV_(Ind)(GLM)-Gag and rVSV_(NJ)(GLM)-Gag. Six (6) week old Balb/cmice, 6 mice/group, were vaccinated with rVSV_(Ind)(GLM)-Gag for primingand rVSV_(NJ)(GLM)-Gag or rVSV_(NJ)(GM)-Gag for booster. Because therVSV_(NJ)(GM) was as attenuated as the rVSV_(NJ)(GLM) in vitro and invivo, the inventors included rVSV_(NJ)(GM)-Gag as a booster virus, andcompared the immune responses to that induced by rVSV_(NJ)(GLM)-Gag.Mice groups were vaccinated with various doses of rVSV_(Ind)(GLM)-Gag,rVSV_(NJ)(GLM)-Gag, and rVSV_(NJ)(GLM)-Gag; 5×10⁶ PFU/mouse, 5×10⁷PFU/mouse, 5×10⁸ PFU/mouse, and 5×10⁹ PFU/mouse (Table 1).

Mice were vaccinated according to the schedule as seen in FIG. 14 andwere sacrificed a week after booster vaccination for splenocytes andserum. The splenocytes were double stained with FITC-anti-CD8 andAPC-anti-IFN-γ for CD8+ T cells with the increased intracellular INF-γ.In order to examine the HIV-1 Gag protein specific CD8+ T cell immuneresponse, splenocytes were isolated and 1×10⁶ cells were stimulated withH-2Kd-restricted HIV-1 Gag peptide, NH2-AMQMLKETI-COOH (SEQ ID NO: 33),the cells were stained with FITC rat anti-mouse CD8 for the CD8molecules and stained with APC rat anti-mouse IFN-γ for intracellularIFN-γ. The secretion of IFN-γ was blocked with Brefeldin A beforestaining them. VSV specific CD8+ T cell immune responses were examinedwith the use of nucleocapsid specific peptide, IN275: NH2-MPYLIDFGL-COOH(SEQ ID NO: 32). Peptide specific CD8+-IFN-γ+ T cells were counted withFACSCalibur, a flowcytometer.

CD8+ T cell immune responses against VSV N protein were very similar inall vaccination groups with different doses of rVSV_(Ind)(GLM)-Gag,rVSV_(NJ)(GLM)-Gag, or rVSV_(NJ)(GM)-Gag (FIG. 19). The HIV-1 Gagspecific CD8+ T cell immune responses after prime vaccination withrVSV_(Ind)(GLM)-Gag and booster with rVSV_(NJ)(GLM)-Gag looked increaseda little bit when the vaccine doses were increased, but differences werenot statistically significant (FIG. 20, groups 2, 3, 4, 5). However,when mice were prime vaccinated with rVSV_(Ind)(GLM)-Gag and boostervaccinated with rVSV_(NJ)(GM)-Gag, the frequency of the HIV-1 Gagspecific CD8+ T cells were increased significantly with the increasingdose of 5×10⁸ PFU. The HIV-1 Gag specific CD8+ T cell response wasstrongest in a group vaccinated with 5×10⁹ PFU/mouse (FIG. 20. groups 6,7, 8, 9). P values on the groups 8 and 9 in FIG. 20 were computed byusing two-sided independent sample t test, the result of group 8 wascompared to that for the group 7, and the result of group 9 was comparedto that for the group 8. The error bars in FIGS. 19 and 20 representstandard error of the mean.

Generation of antibody against HIV-1 Gag protein was increased with theincreasing doses of rVSV_(Ind)(GLM)-Gag for priming and booster withrVSV_(NJ)(GLM)-Gag or rVSV_(NJ)(GM)-Gag (FIG. 21). Booster vaccinationwith rVSV_(NJ)(GLM)-Gag or with rVSV_(NJ)(GM)-Gag generated similarlevel of Gag specific antibodies in various doses of the virus. Theerror bars in FIG. 21 represents standard error of the mean.

Example 13 Generation of rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) ExpressingHIV-1 Proteins; Gag, Pol, Env, and RT

For HIV-1 vaccines utilizing the rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) asvaccine vectors, the inventors included HIV genes encoding most of thelarge polyproteins, which are cleaved into functional proteins-Gag andEnv and proteins with enzymatic activities (pol gene products). Inaddition, HIV-1 gag gene was linked to nucleotides encoding peptideepitopes for T cells and B cells in humans. The inventors have generatedrVSV_(Ind)(GLM) and rVSV_(NJ)(GM) carrying cassettes encoding HIV-1 Gaglinked to B-cell epitopes from HIV Env protein derived from multipleviral clades (FIG. 22). The inventors have generated therVSV_(Ind)(GLM)-Gag-En and rVSV_(NJ)(GM)-Gag-En, which expresses gaggene and T cell peptide epitopes from Nef, Gp120, and Gp41 (FIG. 23).The inventors generated rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) with HIV-1Gag-RT, which encodes gag protein with peptide epitopes from RT, Tat,and Rev proteins (FIG. 23). In addition, the inventors generatedrVSV_(Ind)(GLM) and rVSV_(NJ)(GM) with pol gene and env gene,respectively (FIG. 23). Previously, the inventors have demonstrated thatreplacement of signal peptide of HIV-1 envelope protein with thehoneybee melittin signal peptide dramatically increased the rates ofGp120 expression, glycosylation, and secretion (Yan Li et al., PNAS, 93:9606-9611, 1996). The increased expression and secretion of Gp120 areexpected to enhance the induction of the T cell and B cell immuneresponses against HIV-1 gp120. Therefore, the inventors generated rVSVswith HIV-1 env gene with melittin signal sequence (gp160 mss). Therecombinant VSVs with the HIV-1 proteins were plaque purified and wereamplified for the stock viruses. The expression of HIV-1 proteins at 31°C. and 37° C. from the mutant VSV was examined by Western blot analysis.The Gag proteins tagged with T cell and B cell epitopes from variousHIV-1 proteins were expressed well from rVSV_(Ind)(GLM) andrVSV_(NJ)(GM) at both 31° C. and 37° C. (FIG. 24 and FIG. 25). Thecloned HIV-1 pol gene in the rVSV genome encodes a polyprotein, which iscleaved into protease (PR, p11), reverse transcriptase (RT, p66/p51),and integrase (IN, p31). The inventors detected two subunits of RTproducts, p66 and p51. The p51 is generated from the p66 by proteolyticcleavage of a fragment of p15 at the carboxy terminus. The RT productsp66 and p51 were expressed equally well from both rVSV_(Ind)(GLM) andrVSV_(NJ)(GM) and at both 31° C. and 37° C. (FIG. 26). The env geneencodes glycoprotein Gp160, which is cleaved into Gp120 of receptorbinding protein and gp41 of fusion inducing cytoplasmic andtransmembrane protein. Gp160 is cleaved by the cellular trans-golgiresident protease furin. The Gp160 expressed in BHK21 cells from therVSV_(Ind)(GLM) and rVSV_(NJ)(GM) was not cleaved to Gp120 and Gp41 orthe cleavage was not very efficient, although the level of expressionwas good at both 31° C. and 37° C. (FIG. 27). Our previous data on theexpression of the Gp160 in BHK21 cells and cleavage into Gp120 and Gp41demonstrated that the processing was not very efficient (Kunyu Wu etal., Journal of General Virol., 90:1135-1140, 2009).

Example 14 Immunization Studies in Mice with the rVSV_(Ind)(GLM) andrVSV_(NJ)(GM) with HIV-1 Proteins, Gag, Gp160 and RT

The preliminary prime-boost immunization studies with new M mutantrVSVs, rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) revealed that priming mice withthe rVSV_(Ind)(GLM) and boosting with rVSV_(NJ)(GM) induced better CD8+T cell immune response (FIG. 17) and humoral immune responses (FIG. 18)against the foreign gene than the other way around. With therVSV_(Ind)(GLM)-Gag priming and rVSV_(NJ)(GM)-Gag boosting, the dosageof 5×10⁸ pfu of each virus induced better Gag specific CD8+ T cellresponses than lower dosages, although the B cell responses against Gagwas similar to that induced with the lower dosages (FIG. 20 and FIG.21). The inventors chose 5×10⁸ pfu as a dosage for immunization studieswith rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) expressing HIV-1 Gag, Gp160, andRT.

The inventors prime immunized Balb/c mice with rVSV_(Ind)(GLM)expressing HIV-1 proteins and boost immunized with rVSV_(NJ)(GM)expressing HIV-1 proteins in three weeks after the priming as describedin the table 6. The inventors examined how the immune responses againstthe individual HIV-1 proteins are induced with individual injection orwith the mixed injection of three different viruses expressing separateproteins. One week after the boost immunization, the inventorssacrificed the immunized mice for their splenocytes and sera. The 1×10⁶splenocytes were stimulated with H-2K^(d)-restricted HIV-1 proteinspecific peptides, which are Gag, Env P18, RT354, RT464, and RT472. Thepeptide sequences are shown in the FIG. 29. The stimulated splenocyteswere double stained for the cell surface CD8 molecules and intracellularIFN-γ of T cells with FITC rat anti-mouse CD8 and APC rat anti-mouseIFN-γ. The regimen, prime-boost immunization with rVSV_(Ind)(GLM) andrVSV_(NJ)(GM) induced peptide specific CD8+ T cell immune responses withdifferent degree depending on the peptides the inventors used for thestimulation. CD8+ T cells against Env peptide stimulated CD8+ T cellsthe most up to 19%-23% of CD8+ T cells on average (FIG. 28, G3 and G5).About 4.5% or 3% of CD8+ T cells were responsive to the Gag protein inmice immunized with rVSV expressing Gag alone (FIG. 28, G2) or withmixed three rVSVs expressing Gag A, Gag B, and Gag C linked with variousB and T cell epitopes of HIV-1 Gp120 and Gp41 (FIG. 29, G6). RT specificCD8+ T cells were generated in about 2% of CD8+ T cells in mice injectedwith rVSV with pol gene (FIG. 28, G4). CD8+ T cell responses againstindividual HIV-1 protein, Gag and RT were stronger when mice wereinjected with single rVSV expressing individual protein (FIG. 28, G2 andG4) than when injected with mixed rVSVs expressing single protein (FIG.28, G5). Env specific CD8+ T cell responses were strongest and weresimilar in both single injection and mixed injections. The CD8+ T cellsresponses against Env peptide epitope are immunodominant in Balb/c mice.Currently, the inventors are uncertain why mixed injections with threedifferent rVSVs expressing Gag, Env, and RT induced comparatively lowerCD8+ T cell responses against VSV N, gag, and RT than with the singlevirus injection. It is possible that the competition of each virus forthe antigen presenting cells in the mixed virus injection lowered theCD8+ T cells againt Gag and RT. Because rVSV is common vector for HIV-1proteins in mice either injected with single rVSV or mixed rVSVs, VSV Nsupposed to induce similar or better CD8+ T cell responses in the mixedinjection. However, the CD8+ T cell responses against VSV N in mice arelower even with the single injections when the Env is expressed (FIG.28, G3) as in the mixed injection (FIG. 28, G5). Therefore, it may bethe result of immune dominance of Env protein in Balb/c mice, whichaffects negatively to the presentation of other proteins to the CD8+ Tcells when Env is expressed together. For the combined immunizations inmice, all three rVSVs were injected at the single site, which may haveresulted in the competition of different proteins for the antigenpresentation in the same antigen presentation cells. The inventors willtest the immunization with the mixed injections at more than oneinjection sites to induce CD8+ T cell responses.

HIV-1 Gag proteins expressed in cells can form virus like particles(VLP) and the VLP can be released from the cells. The released VLPs caninduce humoral immune responses against Gag proteins. HIV-1 env geneencodes glycosylated surface proteins, which are processed throughER-golgi network to be properly folded and cleaved into transmembranesubunit Gp41 and surface unit Gp120. The Gp41 and Gp120 are associatedby non-covalent bond and mature to form a trimer of Gp41 and Gp120heterodimer. The matured trimer of Gp41 and Gp120 are exported to thecell surface. The Gp120 tends to fall off from the cell surface becauseof the weak bondage between Gp41 and Gp120. Therefore, antibody againstGp120 can be induced to the cell surface Gp120 or fallen off Gp120.Antibody titer against HIV-1 Gag protein and Gp120 was determined byELISA. For the Gag antibody the microplate was coated with 125 ng/wellof recombinant p55 (Pierce Biotechnology, RP-4921) and 50 μl of micesera was tested with the dilution of 1:100, 1:200, and 1:400. Gagantibody was produced in mice injected with rVSV Gag-En alone (FIG. 30A,G2) and with mixed viruses rVSV-Gag A+rVSV Gag B+rVSV Gag C (FIG. 30A,G6). For the Gp120 ELISA, 96 well microplate was coated with 250 ng/wellof HIV-1 SF162 Gp140 trimer from clade B (NIH AIDS reagent program,cat#12026). Mice sera were diluted in 1:100, 1:200, and 1:400 and 50 μlof the diluted serum was added to the microplate for the ELISA. Antibodyspecific to Gp120 was generated in mice injected with rVSV-HIV-1 Gp160alone and in mice injected with the mixed viruses expressing eachprotein of Gag, Gp160, and RT (FIG. 30B, G3 and G5). The titer of Gp120antibody was slightly lower, although not statistically significant, inmice injected with mixed viruses than in mice injected with single virusexpressing Gp160.

The newly generated M mutants of rVSVs, rVSV_(Ind)(GLM) andrVSV_(NJ)(GM) induced CD8+ T cell and humoral immune responses againstvarious HIV-1 proteins expressed from the vector after injecting micewith an individual virus expressing single virus or with mixed virusesexpressing various HIV-1 proteins.

Example 15 Generation of rVSV_(Ind)(GLM), rVSV_(NJ)(GM), andrVSV_(NJ)(GLM) with Hepatitis C Virus Structural Proteins

Generally, the humoral immune response is the first line of defencemediated by adaptive immune responses against any pathogens. Althoughhumoral immune responses against HCV and its role in the prevention ofHCV infection is not well studied compared to the HCV specific cellularimmune responses, it is worthy to include vaccines which can induce HCVspecific antibodies. It has been demonstrated that nucleocapsid proteincore and surface glycoproteins E1 and E2 form virus-like particles (VLP)which can be released from the cells (Blanchard, E. et al., J. Virol.76:4073-4079, 2002). In addition, it has been demonstrated that HCVprotein p7, a viroporin forming an ion channel in the ER membrane, takespart in releasing the HCV particles from the infected cell (Steinmann,E. et al., PLoS Pathogens 3:962-972, 2007). Another HCV transmembraneprotein NS4B forms a membranous web structure, which mainly consists ofthe ER membrane. The membranous web formed by NS4B is a microstructurefor the production of progeny HCV. It is not well known what functionsNS4B has in the replication of HCV, but it is appealing to include NS4Binto vaccine candidates simply because of its nature forming amembranous structure of ER in which other HCV proteins, especially Core,E1, E2, p7 are localized. Therefore, including Core, E1, E2, P7, andNS4B together into the vaccine may induce humoral and cellular immuneresponses.

For the HCV structural protein vaccines using the new M mutant rVSVs,the inventors inserted the HCV core gene, E1E2P7 and NS4B genes together(connected by the VSV intergenic junction sequences), and CoreE1E2P7 andNS4B genes together (connected by the VSV intergenic junction sequences)to the junction of VSV G gene and L (FIG. 31). The rVSVs;rVSV_(Ind)(GLM)-FC, rVSV_(Ind)(GLM)-CE1E2P7/NS4B, rVSV_(NJ)(GM)-FC, andrVSV_(NJ)(GLM)-E1E2P7/NS4B were generated by reverse genetics. Theinventors had a trouble to generate the rVSV_(NJ)(GM) with CE1E2P7/NS4Bfrom the plasmid DNA, therefore, the inventors removed the core andcloned E1E2P7/NS4B into the plasmid of rVSV_(NJ)(GLM) and generated therVSV_(NJ)(GLM)-E1E2P7/NS4B. The expression of Core, E1, E2, and NS4Bfrom the recombinant VSV at 31° C. and 37° C. was examined by Westernblot analysis using antibodies against each protein (FIG. 32). Theproteins were expressed similarly in quantity at both temperatures. Core(FIG. 32A), E1 (FIG. 32B), and E2 FIG. 32C) were expressed well from therVSV_(Ind)(GLM)-CE1E2P7/NS4B, but the expression of NB4B was lower thanthe other proteins (FIG. 32D).

Example 16 Generation of rVSV_(Ind)(GLM), rVSV_(NJ)(GM), andrVSV_(NJ)(GLM) with HCV Non-Structural Proteins

The inventors want to target most of the HCV proteins including core,E1, E2, NS3, NS4A, NS4B, NS5A and NS5B proteins in order to induce HCVspecific CD8+ T cell and CD4+ T cell immune responses to multipleproteins. HCV nonstructural (NS) proteins-NS3, NS4A, NS4B, NS5A, andNS5B cover more than half of the HCV polyprotein. The NS proteins arecleaved into individual proteins by NS3 with the help of NS4A. Severalstudies demonstrate that patients who recover from the acute HCVinfection develope strong CD4+ T cell and CD8+ T cell responses againstmultiple epitopes in the NS3 protein (Diepolder, H. M. J. Virol.71:6011-6019, 1997; Lamonaca, V. et al. Hepatology 30:1088-1098, 1999;Shoukry, N. H. et al. J. Immunol. 172:483-492, 2004) indicating thatincluding NS3 in the vaccine candidate is important to elicit successfulcellular immune responses against HCV. NS3 protein, a serine proteaseand RNA helicase associates with NS4A and resides on the ER membrane(Sato, S. et al. J. Virol. 69:4255-4260, 1995; Failla, C. et al. J.Virol. 69:1769-1777, 1995). NS5A protein, a phosphoprotein is believedto be involved in HCV RNA synthesis together with NS5B protein, a RNAdependent RNA polymerase (Shirota, Y. et al., J. Biol. Chem.,277:11149-11155, 2002; Shimakami, et al. J. Virol. 78:2738-2748, 2004).NS5B protein is a RNA dependent RNA polymerase that synthesizes positivesense HCV genomic RNA as well as intermediate negative sense genomic RNA(Beherens, S. E. EMBO J. 15:12-22, 1996). NS5B protein is atail-anchored protein and associates with ER membrane through itscarboxyl terminal 20 amino acids (Yamashita, T. J. Biol. Chem.273:15479-15486, 1998; Hagedorn, C. H. Curr. Top. Microbiol. Immunol.242:225-260, 2000).

The inventors cloned HCV NS genes as a gene for a single protein or agene for a polyprotein of 2 or 3 NS proteins. The NS genes NS3, NS34AB,NS5A, NS5B, NS5AB were cloned into the junction at G gene and L gene ofpVSV_(Ind)(GLM) and pVSV_(NJ)(GM) (FIG. 33). The inventors generatedrVSV_(Ind)(GLM)-NS3, rVSV_(Ind)(GLM)-NS34AB, rVSV_(Ind)(GLM)-NS5A,rVSV_(Ind)(GLM)-NS5B, and rVSV_(Ind)(GLM)-NS5AB, and the expression ofthe NS proteins at 31° C. and 37° C. were determined by Western blotanalysis using antibodies against each NS protein (FIG. 34). Theinventors generated rVSV_(NJ)(GM)-NS3, rVSV_(NJ)(GM)-NS4B,rVSV_(NJ)(GM)-NS34AB, rVSV_(NJ)(GM)-NS5A, rVSV_(NJ)(GM)-NS5B, andrVSV_(NJ)(GM)-NS5AB. The expression of each protein from the rVSV_(NJ)vector at 37° C. was examined by Western blot analysis using antibodiesagainst each NS protein (FIG. 35). Although the expression level of NSproteins from the rVSV_(Ind) and rVSV_(NJ) was different, it was goodenough to use them as HCV vaccine.

Example 17 Introduction of Mutations into the M Genes of rVSV_(Ind) andrVSV_(NJ) and Recovery of Recombinant VSV by Reverse Genetics

Mutations were introduced into the M gene of VSV_(Ind) and VSV_(NJ).Nucleotide sequences encoding the amino acids at each position weremutated by the mega-primer PCR method. Each mutation is expressed as asubstitution of an amino acid at a specific position (e.g., M51 in M51R)with another amino acid (e.g., R in M51R). In order to attenuate furtherthe virulence of VSV, the inventors combined mutations (G21E) in thetsO23 with methionine to arginine mutations (M51R) and L111A in the Mgene, which reduced inhibitory activity of M protein on the cellularprotein synthesis, in addition, reduced the assembly of the VSVparticles at non-permissive (39° C.) and semi-permissive (37° C.)temperatures. The nucleotide sequences and amino acid changes from wildtype to mutants in the M genes of rVSV_(Ind) and rVSV_(NJ) are shown inTables 2, 3, 4 and 5. The changed nucleotide codons are underlined andchanged nucleotide sequences and amino acid sequences are bold-faced.

Wild type and mutant recombinant vesicular stomatitis viruses (rVSV)were recovered from the cDNA plasmids by reverse genetics. The VSVreverse genetics employs the BHK21 cells expressing DNA dependant RNApolymerase of bacteriophage T7 (T7) and a plasmid which encodes fulllength genomic RNA of VSV (pVSV) and 3 plasmids expressing nucleocapsidprotein (pN), phosphoprotein (pP), and VSV polymerase L protein (pL).The transcription of the full length genomic RNA and the messenger RNAsfor N, P, and L proteins are under the control of T7 RNA polymerase.Internal ribosome entry site (IRES) at the upstream of each VSV N, P,and L gene enhances the translation of proteins. The plasmids aretransfected into BHK-T7 cells with Lipofectamine™ 2000 in concentrationsof 10 μg of pN, 10 μg of pP, μ5 g of pL, and 15 μg of pVSV. The culturemedia from the transfected cells were harvested when the cells showedabout 50-70% of CPE.

FIG. 36 illustrates the construction of a cDNA plasmid for the fulllength genomic RNA of rVSV_(Ind)(GLM)-new.

First, AGG of M51R in the M gene of prVSV_(Ind)(GLM) was changed to CGAby megaprimer method. The primers, Ind(GLM) M (F) and Ind (GLM) M(M51R)was used to amplify the part of M gene with the additional nucleotidechanges by polymerase chain reaction. The first PCR product was used asa megaprimer together with Ind(GLM) M (R) to amplify the whole M genewith the nucleotide changes, and the second PCR product was cloned intopBluescript II KS vector (Invitrogen) at the restriction site Not I.After confirmation of the nucleotide changes in the M gene clone withthe CGA at the R51, the additional nucleotide change GCA for the Alaninewas introduced by megaprimer PCR method using the primers Ind(GLM) M(F)and Ind(GLM) M(L111A), and Ind(GLM) M(R). The PCR product was clonedagain into the pBluescript II KS vector at the Not I site. The M genewith the nucleotide changes of GAA(E)/CGA(R)/GCA(A) was cut from thepBluescript II KS vector with restriction enzymes, Pac I and Not I andcloned into the prVSV_(Ind)(GLM), which M gene was removed by cuttingwith Pac I and Not I.

Primers: Ind(GLM) M (F);  [SEQ ID NO: 11]5′-cgggcggccgcttaattaaactatgaaaaaaagtaacagat-3′ Ind(GLM) M (M51R); [SEQ ID NO: 12] 5′-catgagtgtctcgctcgtcaac-3′ Ind(GLM) M (L111A); [SEQ ID NO: 13] 5′-aagatcttggcttttgcaggttcttc-3′ Ind(GLM) M (R); [SEQ ID NO: 14] 5′-cgggcggccgctagactagctcatttg-3′

FIG. 37 illustrates the construction of a cDNA plasmid for the fulllength genomic RNA of rVSV_(NJ)(GMM)-new

The nucleotides for the M48R and M51R in the M gene of rVSV_(NJ) waschanged by the megaprimer PCR method. The part of M gene with thenucleotide changes were amplified with the primers, NJ-M(F) andNJ-M(M48R+M51R), and the PCR product was used as a megaprimer with theprimer NJ-G(R) to amplify the full length NJ M gene and G gene in orderto use the restriction enzyme site, Sac I. The M and G PCR product wascut with Pac I and replaced the M and G gene of prVSV_(NJ)(GMM).

Primers: NJ-M (F);  [SEQ ID NO: 15]5′-tccccgcggttaattaagatgaacgatatgaaaaaaact-3′ NJ-M(M48R + M51R); [SEQ ID NO: 16] 5′-tatcatataaatctcgatcctctcgtccgaagaagtca-3′ NJ-G (R); [SEQ ID NO: 17] 5′-tccccgcggttaattaatttagcggaagtgagccat-3′

FIG. 38 shows that rVSV_(Ind)(GLM)-new (G21E/L111A/M51R) with additionalnucleotide changes shows the same temperature sensitivity as originalrVSV_(Ind)(GLM) (G21E/L111F/M51R).

The recovered viruses were purified 3 times by plaque picking and wereamplified for a larger volume of stock viruses by infecting BHK21 cellswith an MOI of 0.1 at 31° C. The inventors infected BHK21 cells with anMOI of 0.1 of rVSV_(Ind)(GLM) and rVSV_(Ind)(GLM)-New. The infectedcells were incubated at permissive temperature (31° C.) andsemi-permissive temperature (37° C., body temperature) to determine thetemperature sensitivity of the new M mutants in the assembly of virusparticles. Culture media from the infected cells were collected at 20hrs post-infection. The number of infectious viral particles in theculture media was determined by plaque assay with Vero E6 cells. Thecells infected with the mutant viruses for the plaque assay wereincubated at 31° C. Both rVSV_(Ind)(GLM) and rVSV_(Ind)(GLM)-New showedthe same temperature sensitivity at 37° C. indicating that that theadditional nucleotide changes in the M gene did not alter thetemperature sensitivity of the rVSV_(Ind)(GLM). The mutation L111A showsthe same temperature sensitivity as L111F indicating that the mutationL111A is not a silent mutation.

The inventors further demonstrated the stability of the new rVSV vectorafter 20 passages at 37 C (about body temperature) and at 31 C (thetemperature for virus amplification) (data not shown). The new GLMmutant (G21E/L111A/M51R) with further nucleotide changes (codons gca forA and cga for R) did not change back to wild type sequences (reversion)after 20 passages in contrast to the old GLM mutant (G21E/L110F/M51R).The result demonstrated that the new GLM mutant is more stable and saferto use for vaccination in humans and other animals.

TABLE 1 Mouse Vaccination Groups and Regimen Prime Boost VaccinationTiter Titer Groups Viruses (PFU)/50 μl Viruses (PFU)/50 μl # of Mice 1Ind (WT)-Gag 5 × 10⁶ NJ (WT)-Gag 5 × 10⁶ 6 2 Ind (GLM)-Gag 5 × 10⁶ NJ(GLM)-Gag 5 × 10⁶ 6 3 Ind (GLM)-Gag 5 × 10⁷ NJ (GLM)-Gag 5 × 10⁷ 6 4 Ind(GLM)-Gag 5 × 10⁸ NJ (GLM)-Gag 5 × 10⁸ 6 5 Ind (GLM)-Gag 5 × 10⁹ NJ(GLM)-Gag 5 × 10⁹ 6 6 Ind (GLM)-Gag 5 × 10⁶ NJ (GM)-Gag 5 × 10⁶ 6 7 Ind(GLM)-Gag 5 × 10⁷ NJ (GM)-Gag 5 × 10⁷ 6 8 Ind (GLM)-Gag 5 × 10⁸ NJ(GM)-Gag 5 × 10⁸ 6 9 Ind (GLM)-Gag 5 × 10⁹ NJ (GM)-Gag 5 × 10⁹ 6 10  NJ(WT)-Gag 5 × 10⁶ Ind (WT)-Gag 5 × 10⁶ 6 Total Mice 60

TABLE 2 Neucleotide Sequence Comparison between M Genes of VSV Indianaserotype, Wild Type (SEQ ID NO: 1) and a Mutant G21E/L111A/MS1R (SEQ ID NO 2)              1                                                   50SEQ ID NO: 1: ATGAGTTCCT TAAAGAAGAT TCTCGGTCTG AAGGGGAAAG GTAAGAAATCSEQ ID NO: 2: ATGAGTTCCT TAAAGAAGAT TCTCGGTCTG AAGGGGAAAG GTAAGAAATC              51       100 SEQ ID NO: 1: TAAGAAATTA GGGATCGCAC CACCCCCTTA TGAAGAGGAC ACTAACATGG SEQ ID NO: 2: TAAGAAATTA GAAATCGCAC CACCCCCTTA TGAAGAGGAC ACTAACATGG               101 150SEQ ID NO: 1: AGTATGCTCC GAGCGCTCCA ATTGACAAAT CCTATTTTGG AGTTGACGAGSEQ ID NO: 2: AGTATGCTCC GAGCGCTCCA ATTGACAAAT CCTATTTTGG AGTTGACGAG              151 200SEQ ID NO: 1: ATGGACACTC ATGATCCGCA TCAATTAAGA TATGAGAAAT TCTTCTTTACSEQ ID NO: 2: CGAGACACTC ATGATCCGCA TCAATTAAGA TATGAGAAAT TCTTCTTTAC              201 250SEQ ID NO: 1: AGTGAAAATG ACGGTTAGAT CTAATCGTCC GTTCAGAACA TACTCAGATGSEQ ID NO: 2: AGTGAAAATG ACGGTTAGAT CTAATCGTCC GTTCAGAACA TACTCAGATG              251 300SEQ ID NO: 1: TGGCAGCCGC TGTATCCCAT TGGGATCACA TGTACATCGG AATGGCAGGGSEQ ID NO: 2: TGGCAGCCGC TGTATCCCAT TGGGATCACA TGTACATCGG AATGGCAGGG              301 350 SEQ ID NO: 1: AAACGTCCCT TCTACAAGAT CTTGGCTTTT TTGGGTTCTT CTAATCTAAASEQ ID NO: 2: AAACGTCCCT TCTACAAGAT CTTGGCTTTT GCAGGTTCTT CTAATCTAAA              351 400SEQ ID NO: 1: GGCCACTCCA GCGGTATTGG CAGATCAAGG TCAACCAGAG TATCACGCTCSEQ ID NO: 2: GGCCACTCCA GCGGTATTGG CAGATCAAGG TCAACCAGAG TATCACGCTC              401 450SEQ ID NO: 1: ACTGTGAAGG CAGGGCTTAT TTGCCACACA GAATGGGGAA GACCCCTCCCSEQ ID NO: 2: ACTGTGAAGG CAGGGCTTAT TTGCCACACA GAATGGGGAA GACCCCTCCC              451 500SEQ ID NO: 1: ATGCTCAATG TACCAGAGCA CTTCAGAAGA CCATTCAATA TAGGTCTTTASEQ ID NO: 2: ATGCTCAATG TACCAGAGCA CTTCAGAAGA CCATTCAATA TAGGTCTTTA              501 550 SEQ ID NO: 1: CAAGGGAACG GTTGAGCTCA CAATGACCAT CTACGATGAT GAGTCACTGGSEQ ID NO: 2: CAAGGGAACG GTTGAGCTCA CAATGACCAT CTACGATGAT GAGTCACTGG              551 600SEQ ID NO: 1: AAGCAGCTCC TATGATCTGG GATCATTTCA ATTCTTCCAA ATTTTCTGATSEQ ID NO: 2: AAGCAGCTCC TATGATCTGG GATCATTTCA ATTCTTCCAA ATTTTCTGAT              601 650SEQ ID NO: 1: TTCAGAGATA AGGCCTTAAT GTTTGGCCTG ATTGTCGAGA AAAAGGCATCSEQ ID NO: 2: TTCAGAGATA AGGCCTTAAT GTTTGGCCTG ATTGTCGAGA AAAAGGCATC              651 700 SEQ ID NO: 1: TGGAGCTTGG GTCCTGGATT CTGTCAGCCA CTTCAAATGASEQ ID NO: 2: TGGAGCTTGG GTCCTGGATT CTGTCAGCCA CTTCAAATGA

TABLE 3 Amino Acid Sequence Comparison between M Proteins of VSVIndiana serotype Wild Type (SEQ ID NO: 3) and a MutantG21E/L111A/M51R (SEQ ID NO: 4)              1                    21                             50SEQ ID NO: 3: MSSLKKILGL KGKGKKSKKL GIAPPPYEED TNMEYAPSAP IDKSYFGVDESEQ ID NO: 4: MSSLKKILGL KGKGKKSKKL EIAPPPYEED TNMEYAPSAP IDKSYFGVDE              51 100SEQ ID NO: 3: MDTHDPHQLR YEKFFFTVKM TVRSNRPFRT YSDVAAAVSH WDHMYIGMAGSEQ ID NO: 4: RDTHDPHQLR YEKFFFTVKM TVRSNRPFRT YSDVAAAVSH WDHMYIGMAG              101       111 150SEQ ID NO: 3: KRPFYKILAF LGSSNLKATP AVLADQGQPE YHAHCEGRAY LPHRMGKTPPSEQ ID NO: 4: KRPFYKILAF AGSSNLKATP AVLADQGQPE YHAHCEGRAY LPHRMGKTPP              151 200SEQ ID NO: 3: MLNVPEHFRR PFNIGLYKGT VELTMTIYDD ESLEAAPMIW DHFNSSKFSDSEQ ID NO: 4: MLNVPEHFRR PFNIGLYKGT VELTMTIYDD ESLEAAPMIW DHFNSSKFSD              201                           229 250SEQ ID NO: 3: FRDKALMFGL IVEKKASGAW VLDSVSHFKSEQ ID NO: 4: FRDKALMFGL IVEKKASGAW VLDSVSHFK

TABLE 4 Nucleotide Sequence Comparison between M Genes of VSV NewJersey serotype Wild Type (SEQ ID NO: 5) and Mutants, G22E/M48R/M5lIt(SEQ ID NO: 6) and G22E/L110A/M48R/M51R (SEQ ID NO: 7)              1                                                   50SEQ ID NO: 5: ATGAGTTCCT TCAAAAAGAT TCTGGGATTT TCTTCAAAAA GTCACAAGAASEQ ID NO: 6: ATGAGTTCCT TCAAAAAGAT TCTGGGATTT TCTTCAAAAA GTCACAAGAASEQ ID NO: 7: ATGAGTTCCT TCAAAAAGAT TCTGGGATTT TCTTCAAAAA GTCACAAGAA              51                                                   100SEQ ID NO: 5: ATCAAAGAAA CTAGGC TTGC CACCTCCTTA TGAGGAATCA AGTCCTATGGSEQ ID NO: 6: ATCAAAGAAA CTAGAA TTGC CACCTCCTTA TGAGGAATCA AGTCCTATGGSEQ ID NO: 7: ATCAAAGAAA CTAGAA TTGC CACCTCCTTA TGAGGAATCA AGTCCTATGG              101                                                  150SEQ ID NO: 5: AGATTCAACC ATCTGCCCCA TTATCAAATG ACTTCTTCGG AATG GAGGATSEQ ID NO: 6: AGATTCAACC ATCTGCCCCA TTATCAAATG ACTTCTTCGG ACGAGAGGATSEQ ID NO: 7: AGATTCAACC ATCTGCCCCA TTATCAAATG ACTTCTTCGG ACGAGAGGAT              151                                                  200SEQ ID NO: 5: ATG GATTTAT ATGATAAGGA CTCCTTGAGA TATGAGAAGT TCCGCTTTATSEQ ID NO: 6: CGAGATTTAT ATGATAAGGA CTCCTTGAGA TATGAGAAGT TCCGCTTTATSEQ ID NO: 7: CGAGATTTAT ATGATAAGGA CTCCTTGAGA TATGAGAAGT TCCGCTTTAT              201                                                  250SEQ ID NO: 5: GTTGAAGATG ACTGTTAGAG CTAACAAGCC CTTCAGATCG TATGATGATGSEQ ID NO: 6: GTTGAAGATG ACTGTTAGAG CTAACAAGCC CTTCAGATCG TATGATGATGSEQ ID NO: 7: GTTGAAGATG ACTGTTAGAG CTAACAAGCC CTTCAGATCG TATGATGATG              251                                                  300SEQ ID NO: 5: TCACCGCAGC GGTATCACAA TGGGATAATT CATACATTGG AATGGTTGGASEQ ID NO: 6: TCACCGCAGC GGTATCACAA TGGGATAATT CATACATTGG AATGGTTGGASEQ ID NO: 7: TCACCGCAGC GGTATCACAA TGGGATAATT CATACATTGG AATGGTTGGA              301                                                  350SEQ ID NO: 5: AAGCGTCCTT TCTACAAGAT AATTGCTCTG  ATTGGCTCCA GTCATCTGCASEQ ID NO: 6: AAGCGTCCTT TCTACAAGAT AATTGCTCTG  ATTGGCTCCA GTCATCTGCASEQ ID NO: 7: AAGCGTCCTT TCTACAAGAT AATTGCTGCA ATTGGCTCCA GTCATCTGCA              351                                                  400SEQ ID NO: 5: AGCAACTCCA GCTGTGTTGG CAGACTTAAA TCAACCAGAG TATTATGCCASEQ ID NO: 6: AGCAACTCCA GCTGTGTTGG CAGACTTAAA TCAACCAGAG TATTATGCCASEQ ID NO: 7: AGCAACTCCA GCTGTGTTGG CAGACTTAAA TCAACCAGAG TATTATGCCA              401                                                  450SEQ ID NO: 5: CACTAACAGG TCGTTGTTTT CTTCCTCACC GACTCGGATT GATCCCACCGSEQ ID NO: 6: CACTAACAGG TCGTTGTTTT CTTCCTCACC GACTCGGATT GATCCCACCGSEQ ID NO: 7: CACTAACAGG TCGTTGTTTT CTTCCTCACC GACTCGGATT GATCCCACCG              451                                                  500SEQ ID NO: 5: ATGTTTAATG TGTCCGAAAC TTTCAGAAAA CCATTCAATA TTGGGATATASEQ ID NO: 6: ATGTTTAATG TGTCCGAAAC TTTCAGAAAA CCATTCAATA TTGGGATATASEQ ID NO: 7: ATGTTTAATG TGTCCGAAAC TTTCAGAAAA CCATTCAATA TTGGGATATA              501                                                 550SEQ ID NO: 5: CAAAGGGACT CTCGACTTCA CCTTTACAGT TTCAGATGAT GAGTCTAATGSEQ ID NO: 6: CAAAGGGACT CTCGACTTCA CCTTTACAGT TTCAGATGAT GAGTCTAATGSEQ ID NO: 7: CAAAGGGACT CTCGACTTCA CCTTTACAGT TTCAGATGAT GAGTCTAATG              551                                                  600SEQ ID NO: 5: AAAAAGTCCC TCATGTTTGG GAATACATGA ACCCAAAATA TCAATCTCAGSEQ ID NO: 6: AAAAAGTCCC TCATGTTTGG GAATACATGA ACCCAAAATA TCAATCTCAGSEQ ID NO: 7: AAAAAGTCCC TCATGTTTGG GAATACATGA ACCCAAAATA TCAATCTCAG              601                                                  650SEQ ID NO: 5: ATCCAAAAAG AAGGGCTTAA ATTCGGATTG ATTTTAAGCA AGAAAGCAACSEQ ID NO: 6: ATCCAAAAAG AAGGGCTTAA ATTCGGATTG ATTTTAAGCA AGAAAGCAACSEQ ID NO: 7: ATCCAAAAAG AAGGGCTTAA ATTCGGATTG ATTTTAAGCA AGAAAGCAAC              651                                                  700SEQ ID NO: 5: GGGAACTTGG GTGTTAGACC AATTGAGTCC GTTTAASEQ ID NO: 6: GGGAACTTGG GTGTTAGACC AATTGAGTCC GTTTAASEQ ID NO: 7: GGGAACTTGG GTGTTAGACC AATTGAGTCC GTTTAA

TABLE 5 Amino Acid Sequence Comparison between M Proteins of VSV NewJersey serotype Wild Type (SEQ ID NO: 8) and Mutants,G22E/M48R/M5lIt(SEQ ID NO: 9) and G22E /L110A/M48R/M51R (SEQ ID NO: 10)              1                      22                         48 50SEQ ID NO: 8 :MSSFKKILGF SSKSHKKSKK LGLPPPYEES SPMEIQPSAP LSNDFFGMEDSEQ ID NO: 9: MSSFKKILGF SSKSHKKSKK LELPPPYEES SPMEIQPSAP LSNDFFGREDSEQ ID NO: 10:MSSFKKILGF SSKSHKKSKK LELPPPYEES SPMEIQPSAP LSNDFFGRED              51                                                  100SEQ ID NO: 8: MDLYDKDSLR YEKFRFMLKM TVRANKPFRS YDDVTAAVSQ WDNSYIGMVGSEQ ID NO: 9: RDLYDKDSLR YEKFRFMLKM TVRANKPFRS YDDVTAAVSQ WDNSYIGMVGSEQ ID NO: 10:RDLYDKDSLR YEKFRFMLKM TVRANKPFRS YDDVTAAVSQ WDNSYIGMVG              101     110                                         150SEQ ID NO: 8: KRPFYKIIAL IGSSHLQATP AVLADLNQPE YYATLTGRCF LPHRLGLIPPSEQ ID NO: 9: KRPFYKIIAL IGSSHLQATP AVLADLNQPE YYATLTGRCF LPHRLGLIPPSEQ ID NO: 10:KRPFYKIIAA IGSSHLQATP AVLADLNQPE YYATLTGRCF LPHRLGLIPP              151                                                  200SEQ ID NO: 8: MFNVSETFRK PFNIGIYKGT LDFTFTVSDD ESNEKVPHVW EYMNPKYQSQSEQ ID NO: 9: MFNVSETFRK PFNIGIYKGT LDFTFTVSDD ESNEKVPHVW EYMNPKYQSQSEQ ID NO: 10:MFNVSETFRK PFNIGIYKGT LDFTFTVSDD ESNEKVPHVW EYMNPKYQSQ              201                                                  250SEQ ID NO: 8: IQKEGLKFGL ILSKKATGTW VLDQLSPFKSEQ ID NO: 9: IQKEGLKFGL ILSKKATGTW VLDQLSPFKSEQ ID NO: 10:IQKEGLKFGL ILSKKATGTW VLDQLSPFK

TABLE 6 Vaccination studies in mice for broad range CD8+ T cellresponses and humoral immune responses against HIV-1 proteins Vacci-nation rVSV w/ HIV-1 Proteins Groups Prime Boost G1 rVSV_(Ind)(GLM)rVSV_(NJ)(GM) G2 rVSV_(Ind)(GLM)-HIV-1 Gag-En rVSV_(NJ)(GM)-HIV-1 Gag-EnG3 rVSV_(Ind)(GLM)-HIV-1 rVSV_(NJ)(GM)-HIV-1 Gp160mss Gp160mss G4rVSV_(Ind)(GLM)-HIV-1 Pol rVSV_(NJ)(GM)-HIV-1 Pol G5rVSV_(Ind)(GLM)-HIV-1 Gag-En + rVSV_(NJ)(GM)-HIV-1rVSV_(Ind)(GLM)-Gp160mss + Gag-En + rVSV_(Ind)(GLM)-HIV-1 PolrVSV_(NJ)(GM)-Gp160mss + rVSV_(NJ)(GM)-HIV1 Pol G6 rVSV_(Ind)(GLM)-HIV-1Gag-A + rVSV_(NJ)(GM)-HIV-1 Gag-A + rVSV_(Ind)(GLM)-HIV-1 Gag-B +rVSV_(NJ)(GM)-HIV-1 Gag-B + rVSV_(Ind)(GLM)-HIV-1 Gag-CrVSV_(NJ)(GM)-HIV-1 Gag-C

What is claimed is:
 1. A modified matrix (M) protein of a vesicularstomatitis virus (VSV), wherein the modified M protein comprises anamino acid sequence selected from: (i) SEQ ID NO: 3 including at leastthe following substitutions: G21E/L111A/M51R; and (ii) SEQ ID NO: 8including at least the following substitutions: G22E/L110A/M48R/M51R. 2.The modified M protein of claim 1, wherein the modified M protein of (i)comprises an amino acid sequence SEQ ID NO:4 and the modified M proteinof (ii) comprises the amino acid sequence SEQ ID NO:10.
 3. The modifiedM protein of claim 1, wherein the E at position 21 in (i) and the E atposition 22 in (ii) are encoded by a gaa codon, and wherein the R atposition 51 in (i) and (ii) and the R at position 48 in (ii) is encodedby a cga codon.
 4. The modified M protein of claim 1, wherein the A atposition 111 in (i) and the A at position 110 in (ii) is encoded by agca codon.
 5. The modified M protein of claim 1, wherein the amino acidsequence of (i) is encoded by a gene comprising SEQ ID NO:2, and theamino acid sequence of (ii) is encoded by a gene comprising SEQ ID NO:7.6. A nucleotide sequence that encodes for a modified matrix protein of avesicular stomatitis virus, wherein the nucleotide sequence comprises asequence selected from: SEQ ID NO:2, SEQ ID NO:6 and SEQ ID NO:7.
 7. Arecombinant vesicular stomatitis virus (rVSV), wherein said rVSVincludes a nucleotide sequence that encodes for a modified matrix (M)protein, the nucleotide sequence comprising a sequence selected from SEQID NO:2, SEQ ID NO:6 and SEQ ID NO:7.
 8. A recombinant vesicularstomatitis virus (rVSV), wherein said rVSV comprises a modified matrix(M) protein, the modified M protein comprising an amino acid sequenceselected from: (i) SEQ ID NO: 3 including at least the followingsubstitutions: G21E/L111A/M51R; and (ii) SEQ ID NO: 8 including at leastthe following substitutions: G22E/L110A/M48R/M51R.
 9. The rVSV of claim7, wherein the E at position 21 in (i) and the E at position 22 in (ii)are encoded by a gaa codon, and wherein the R at position 51 in (i) and(ii) and the R at position 48 in (ii) is encoded by a cga codon.
 10. TherVSV of claim 7, wherein the A at position 111 in (i) and the A atposition 110 in (ii) are encoded by a gca codon.
 11. The rVSV of claim7, wherein said rVSV is a recombinant vesicular stomatitis virus Indianaserotype (rVSV_(Ind)), and wherein the modified M protein comprises theamino acid sequence of SEQ ID NO: 3 including at least the followingsubstitution: G21E/L111A/M51R.
 12. The rVSV of claim 10, wherein themodified M protein comprises the amino acid sequence of SEQ ID NO: 4.13. The rVSV of claim 10, wherein the modified M protein is encoded by agene comprising a nucleotide sequence of SEQ ID NO:
 2. 14. The rVSVaccording to any one of claims 10-12, wherein the rVSV_(Ind) is capableof producing VSV_(Ind) particles at permissible temperatures andincapable of producing the particles at non-permissible temperatures.15. The rVSV of claim 7, wherein said rVSV is a recombinant vesicularstomatitis virus New Jersey serotype (rVSV_(NJ)), and wherein themodified M protein comprises the amino acid sequence of SEQ ID NO: 8including at least the following substitutions: G22E/L110A/M48R/M51R.16. The rVSV of claim 14, wherein the modified M protein is encoded by agene comprising a nucleotide sequence of SEQ ID NO: 7
 17. The rVSV ofclaim 14, wherein the modified M protein comprises the amino acidsequence SEQ ID NO:
 10. 18. The rVSV of claims 7-17, wherein the rVSV isa chimeric rVSV that expresses a protein of a foreign pathogen.
 19. TherVSV of claim 17, wherein said pathogen is a viral, fungal, bacterial orparasitic pathogen.
 20. The rVSV of claims 7-19, wherein the rVSV isessentially non-cytolytic and avirulent.
 21. A vaccine, the vaccineincluding a modified matrix (M) protein of a vesicular stomatitis virus(VSV) wherein the modified M protein is encoded by a nucleotide sequencecomprising a sequence selected from: SEQ ID NO:2, SEQ ID NO:6 and SEQ IDNO:7.
 22. A vaccine, wherein the vaccine comprises an effective amountof one or more attenuated recombinant vesicular stomatitis virus (rVSV),the one or more attenuated rVSVs including a modified matrix (M)protein, the modified M protein comprising an amino acid sequenceselected from: (i) SEQ ID NO: 3 including at least the followingsubstitutions: G21E/L111A/M51R, and (ii) SEQ ID NO: 8 including at leastthe following substitutions: G22E/L110A/M48R/M51R.
 23. The vaccine ofclaim 22, wherein the rVSV is a recombinant vesicular stomatitis virusIndiana serotype (rVSV_(Ind)), and wherein the modified M proteincomprises the amino acid sequence of SEQ ID NO: 3 including at least thefollowing substitution: G21E/L111A/M51R.
 24. The vaccine of claim 23,wherein the modified M protein comprises the amino acid sequence of SEQID NO:
 4. 25. The vaccine of claim 23, wherein the modified M protein isencoded by a gene comprising a nucleotide sequence of SEQ ID NO:
 2. 26.The vaccine of any one of claims 23 to 25, wherein the rVSV_(Ind) iscapable of producing VSV_(Ind) particles at permissible temperatures andincapable of producing the particles at non-permissible temperatures.27. The vaccine of claims 23-26, wherein the rVSV_(Ind) is a full lengthrVSV_(Ind).
 28. The vaccine of claim 22, wherein said rVSV is arecombinant vesicular stomatitis virus New Jersey serotype (rVSV_(NJ)),and wherein the modified M protein comprises the amino acid sequence ofSEQ ID NO: 8 including at least the following substitutions:G22E/L110A/M48R/M51R.
 29. The vaccine of claim 28, wherein the modifiedM protein comprises the amino acid sequence SEQ ID NO:
 10. 30. Thevaccine of claim 28, wherein the modified M protein is encoded by a genecomprising a nucleotide sequence of SEQ ID NO:
 7. 31. The vaccine ofclaims 28-30, wherein the rVSV_(NJ) is a full length rVSV_(NJ).
 32. Thevaccine of any one of claims 21 to 31, wherein the rVSV is a chimericrVSV that expresses a protein of a foreign pathogen, and wherein saidchimeric rVSV is capable of inducing an immune response to said protein.33. The vaccine of claim 32, wherein the vaccine comprises a mixture ofattenuated chimeric rVSVs, wherein at least two chimeric rVSVs in themixture express a different protein of the foreign pathogen.
 34. Thevaccine of claim 32, wherein the pathogen is a viral, a fungal, abacterial or a parasitic pathogen.
 35. The vaccine of claim 32, whereinthe pathogen is a lentivirus.
 36. The vaccine according to claim 35,wherein the lentivirus is a HIV and the protein of the foreign pathogenis a HIV protein.
 37. The vaccine of claim 32, wherein the pathogen isHCV and the protein of the foreign pathogen is a HCV protein.
 38. Thevaccine of claim 22, wherein the E at position 21 in (i) and the E atposition 22 in (ii) are encoded by a gaa codon, and wherein the R atposition 51 in (i) and (ii) and the R at position 48 in (ii) is encodedby a cga codon.
 39. The vaccine of claim 22, wherein the A at position111 in (i) and the A at position 110 in (ii) are encoded by a gca codon.40. The vaccine of any one of claims 21 to 39, wherein said vaccine iscapable of inducing a humoral, cellular and mucosal immune response. 41.The vaccine of any one of claims 21 to 40, wherein said vaccine furtherincludes an adjuvant.
 42. A prime boost combination vaccine, wherein theprime boost combination vaccine comprises: (a) an effective amount of avaccine comprising an attenuated recombinant vesicular stomatitis virus(rVSV) of one serotype having a first modified matrix (M) proteincomprising the amino acid sequence of SEQ ID NO:4; and (b) an effectiveamount of a vaccine comprising a rVSV of another serotype having asecond modified M protein comprising the amino acid sequence of SEQ IDNO:9 or SEQ ID NO:10.
 43. The prime boost combination vaccine of claim42, wherein SEQ ID NO:4 is encoded by a gene comprising SEQ ID NO:2. 44.The prime boost combination vaccine of claim 42, wherein SEQ ID NO:9 isencoded by a gene comprising SEQ ID NO:6, and SEQ ID NO:10 is encoded bya gene comprising SEQ ID NO:7.
 45. The prime boost combination vaccineof claim 42, wherein (a) is a priming vaccine and (b) is a boostervaccine.
 46. The prime boost combination vaccine of claim 42, wherein(b) is a priming vaccine and (a) is a booster vaccine.
 47. The primeboost combination vaccine of claim 42, wherein the two attenuated rVSVsare chimeric rVSVs that express a protein of a foreign pathogen, andwherein the two chimeric rVSVs are capable of inducing an immuneresponse to the protein.
 48. The prime boost combination vaccine ofclaim 47, wherein the pathogen is a viral, a fungal, a bacterial or aparasitic pathogen.
 49. The prime boost combination vaccine of claim 47,wherein the pathogen is a lentivirus.
 50. The prime boost combinationvaccine of claim 49, wherein the lentivirus is a HIV and the protein isa HIV protein.
 51. The prime boost combination vaccine of claim 50,wherein the rVSV of one serotype and the rVSV of the other serotypeinclude a surface glycoprotein (G) gene and a RNA dependent RNApolymerase (L) gene, and wherein a gene for expressing the HIV proteinis inserted in between the G gene and the L gene.
 52. The prime boostcombination vaccine of claim 51, wherein the HIV gene is selected fromthe group of HIV genes consisting of env, gag and pol.
 53. The primeboost combination vaccine of claim 49, wherein the pathogen is HCV andthe epitope is a HCV protein.
 54. The prime boost combination vaccine ofclaim 53, wherein the rVSV of one serotype and the rVSV of the otherserotype include a surface glycoprotein (G) gene and a RNA dependent RNApolymerase (L) gene, and wherein a gene for expressing the HCV proteinis inserted in between the G gene and the L gene.
 55. The prime boostcombination vaccine of claim 54, wherein the HCV protein is a structuralor a non-structural HCV protein.
 56. The prime boost combination vaccineof any one of claims 47 to 55, wherein each one of the two vaccinescomprise a mixture of the attenuated chimeric rVSVs, and wherein atleast two of the attenuated chimeric rVSVs in the mixture have adifferent protein of the pathogen.
 57. The prime boost combinationvaccine of any one of claims 42 to 54, wherein each one of the twovaccines is capable of inducing humoral, cellular and mucosal immuneresponses.
 58. The prime boost combination vaccine of any one of claims42 to 57, wherein the serotype of vaccine (a) is Indiana and theserotype of vaccine (b) is New Jersey.
 59. The prime boost combinationvaccine of any one of claims 42 to 58, wherein each one of vaccine (a)and vaccine (b) further comprises an adjuvant.
 60. A kit comprising: (a)at least one dose of an effective amount of a vaccine comprising arecombinant vesicular stomatitis virus Indiana serotype (rVSV_(Ind))having a modified matrix (M) protein comprising the amino acid sequenceof SEQ ID NO:4, and (b) at least one dose of an effective amount of avaccine comprising a recombinant vesicular stomatitis virus New Jerseyserotype (rVSV_(NJ)) having a modified M protein comprising the aminoacid sequence of SEQ ID NO:9 or the amino acid sequence of SEQ ID NO:10.61. The kit of claim 60, wherein (a) and (b) are formulated in apharmaceutically acceptable carrier.
 62. The kit of claim 60, whereinSEQ ID NO:4 is encoded by a gene comprising SEQ ID NO:2.
 63. The kit ofclaim 60, wherein SEQ ID NO:9 is encoded by a gene comprising SEQ IDNO:6 and SEQ ID NO:10 is encoded by a gene comprising SEQ ID NO:7. 64.An isolated peptide comprising an amino acid sequence selected from thegroup of amino acid sequences listed as SEQ ID NOs: 4 and
 10. 65. Anisolated nucleotide sequences comprising a nucleotide sequence selectedfrom the group SEQ ID NO:2, SEQ ID NO:6 and SEQ ID NO:7.
 66. A use ofthe vaccine of claim 21 or 22 to induce an immune response in a subject.67. A use of the prime boost combination vaccine of claim 42 to inducean immune response in a subject.
 68. A method of inducing an immuneresponse in a subject, wherein the method comprises administering to thesubject: (a) an effective amount of a vaccine comprising an attenuatedrecombinant vesicular stomatitis virus (rVSV) of one serotype having afirst modified M protein, the first modified M protein comprising theamino acid sequence of SEQ ID NO: 3 including at least the followingsubstitutions: G21E/L111A/M51R; and (b) an effective amount of anothervaccine comprising an attenuated rVSV of another serotype having asecond modified M protein, the second modified M protein comprising theamino acid sequence of SEQ ID NO: 8 including at least the followingsubstitutions: G22E/L110A/M48R/M51R.
 69. The method of claim 68, wherein(a) is administered to the subject before (b) is administered to thesubject.
 70. The method of claim 69, wherein (b) is administered to thesubject more than one time over the course of inducing.
 71. The methodof claim 68, wherein (a) is administered to the subject and (b) isadministered to the subject at about weeks three, eight and sixteenpost-administration of (a).
 72. The method of claim 68, wherein (b) isadministered to the subject before (a) is administered to the subject.73. The method of claim 68, wherein (a) is administered to the subjectmore than one time over the course of inducing.
 74. The method of claim72, wherein (b) is administered to the subject and (a) is administeredto the subject at about weeks three, eight and sixteenpost-administration of (b).
 75. The method of claim 68, wherein the tworVSVs are chimeric rVSVs that express a protein of a foreign pathogen,and wherein the two rVSVs are capable of inducing an immune response tothe protein.
 76. The method of claim 75, wherein the pathogen is aviral, a fungal, a bacterial or a parasitic pathogen.
 77. The method ofclaim 75, wherein the pathogen is a lentivirus.
 78. The method accordingto claim 77, wherein the lentivirus is a human immunodeficiency virus(HIV) and the protein is a HIV protein.
 79. The method of claim 78,wherein the rVSV of one serotype and the rVSV of the other serotypeinclude a surface glycoprotein (G) gene and a RNA dependent RNApolymerase (L) gene, and wherein a gene for expressing the HIV proteinis a HIV gene inserted in between the G gene and the L gene.
 80. Themethod of claim 79, wherein the HIV gene is selected from the group ofHIV genes consisting of gag, env and pol.
 81. The method of claim 75,wherein the pathogen is hepatitis C virus (HCV) and the protein is a HCVprotein.
 82. The method of claim 81, wherein the rVSV of one serotypeand the rVSV of the other serotype include a surface glycoprotein (G)gene and a RNA dependent RNA polymerase (L) gene, and wherein a gene forexpressing the HCV protein is inserted in between the G gene and the Lgene of the rVSV.
 83. The method of claim 75, wherein the two vaccinescomprise a mixture of attenuated chimeric rVSVs, wherein at least two ofthe attenuated chimeric rVSVs in the mixture express a different proteinof the pathogen.
 84. The method of any one of claims 68 to 83, whereineach one of the two vaccines (a) and (b) induces humoral, cellular andmucosal immune responses.
 85. The method of any one of claims 68 to 84,wherein the one serotype of vaccine (a) is Indiana and the otherserotype of vaccine (b) is New Jersey.
 86. The method of any one ofclaims 68 to 85, wherein each of vaccine (a) and vaccine (b) furthercomprises an adjuvant.